Proteomics study of silver nanoparticles toxicity on Bacillus thuringiensis
Introduction
It has been discovered that silver and its related compounds are effective anti-microbial agents. They have a variety of applications including a prophylactic effect for human infections, the treatment of wounds and on plant and animal microbial diseases (Navarro et al., 2008). Silver nanoparticles (AgNPs) as a newborn form of silver represent a novel generation of anti-microbial, anti-fungal and anti-viral applications against a very broad range of microorganisms (Kim et al., 2008, Rai et al., 2009). Environmental pollution caused by AgNPs, particularly contamination of soil and water resources, has been accelerated as a result of global industrialization and is considered a major risk for communities throughout the world (Navarro et al., 2008).
Soil ecosystems have been exposed to toxic compounds, among which are AgNPs (Filip, 2002). Some of the soil bacteria communities have an outstanding ability to adapt to a polluted environment according to employ various unique mechanisms (Wang et al., 2007). Consequently, polluted soil is faced with new kinds of micro flora; some have changed significantly, others are simply missing. Several negative effects on soil beneficial bacteria, suggesting that they have adverse reactions to pollution such as nanoparticles (Filip, 2002, Wang et al., 2007). Bacillus thuringiensis (Bt) is a Gram-positive soil-dwelling bacterium, commonly used as a biological pesticide. Its cry toxin is extracted and used as a pesticide. Many Bt strains produce crystal proteins (proteinaceous inclusions), called δ-endotoxins, that have insecticidal action. This compound has led to their use as an insecticide, and Bt genes are used to genetically modify crops recently (Höfte and Whiteley, 1989).
A bacterial cell can react simultaneously to a wide variety of stresses (Vollmer et al., 2008). Stress response mechanism of bacteria enables them to survive adverse and mutable conditions in their immediate environments. Various mechanisms have been suggested to bacterial responses following the different environmental fluctuations. The stress response in bacteria involves a number of systems that act against an external stimulus. A complex network of global regulatory systems in bacteria certifies that various stress response systems interact with each other and this leads to a coordinated and effective response.
Proteomics is a fastest developing field of research and it contributes substantially to our understanding of organisms at the cellular level (Lok et al., 2006, Navarro et al., 2008). In addition, this is the study of functions and regulation of biological systems based on analysis of the protein expression profile. Furthermore, there is general agreement that soil bacterial proteomics may be a tool for its better management. Because of the ability of soil to stabilize extracellular proteins over various mechanisms, development of bacterial proteomics needs an assessment of the efficiency of protein extraction from various soil types. In the case of nanotechnology environment proteomics Li et. al. used the gel-base proteomics together with other biochemical studies to evaluate the AgNPs action on Staphylococcus aureus (Li et al., 2011). The aim of this study was to evaluate the action of homemade spherical colloidal AgNPs suspension against Bacillus thuringiensis (isolate from Oryza sativa L. rhizosphere) from proteomic point of view using the gel-base combined NanoLC/FT-ICR MSMS method.
Section snippets
Silver nanoparticles (AgNPs): synthesis and characterization
The homemade silver nanoparticle colloidal suspension (AgNPs) was prepared and characterized according to the previously reported method (Mirzajani et. al, 2011). Briefly, colloidal AgNPs with spherical morphology and distinct diffraction peaks of crystalline plans of cubic including (111), (200), (220) and (311), have a maximum absorbance (λmax) at 426 nm. Their size was 18.34 nm (X99) with the homogeneity of their size within the range of 0.1–1000 nm.
Bacterial identification, cultivation and AgNPs treatment
Rice (O. sativa L.) rhizosphere (the soil
2-DE gel electrophoresis optimization
Proteomes were then analyzed by 2-DE followed by silver staining. It was previously reported that 18 cm IPG strips have the capability to load ≤70–100 µg/gel of proteins. As a result the 2D-page of 100 µg/gel of proteins (0 of AgNPs µg/mL treatment) was prepared to optimize the quality of the gel. As demonstrated in Fig. 1d, the 100 µg/gel amounted to too much loading and the proteins were not efficiently resolved. For this reason the amount of loading should be decreased to get the best
Discussions
The detailed mechanism of AgNPs toxic effect on bacteria is not completely understood. There are some hypotheses like cell wall “pit” formation, oxidative stress following the ROS production and direct binding to proteins (Li et al., 2010). Based on our previous study on S. aureus, it was found that bacteria could survive at low concentrations of AgNPs. Therefore it can be postulated that the structures of the primary cell walls may be affected resulting in the contact with AgNPs (Mirzajani et
Conclusion
To our knowledge, this is the first report on proteome analysis of a B. thuringiensis and its response to AgNPs treatment. Responsive protein changed in terms of its abundance rather than in-gel position or its presence or absence. In conclusion, the above experiments demonstrated the possibility of AgNPs activity on the cell wall. In addition, it is very likely that AgNPs passed the cell wall and interacted with normal cell metabolic processes such as protein synthesis and cell division.
Acknowledgments
Financial support from the Research Council of Shahid Beheshti Universityand the Iran Nanotechnology Initiative Council is gratefully acknowledged. This publication represents a component of the doctoral thesis of Fateme Mirzajani at the Medicinal Plants and Drug Research Institute of Shahid Beheshti University, Tehran, Iran.
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