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Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.

Ravindranath SP, Henne KL, Thompson DK, Irudayaraj J - PLoS ONE (2011)

Bottom Line: Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells.Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported.While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

View Article: PubMed Central - PubMed

Affiliation: Bindley Bioscience Center, Birck Nanotechnology Center, Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States of America. josephi@purdue.edu

ABSTRACT
This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

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Thin-section TEM Images of S. oneidensis MR-1.A. without particles, B. plain 13 nm gold Nanoparticles, Fig. 4C–4D. Chromate coated gold nanoparticles, Cr-AuNp:13 nm, (Fig. 4E–4F) 3.5 nm Cr-AuNp. Red arrows indicate extracellularly bound Cr-AuNp and green arrows/circle indicate internalized particles. (Fig. 4G–4H) show 3.5 nm and 13 nm probes used in Cr-AuNp preparation.
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pone-0016634-g004: Thin-section TEM Images of S. oneidensis MR-1.A. without particles, B. plain 13 nm gold Nanoparticles, Fig. 4C–4D. Chromate coated gold nanoparticles, Cr-AuNp:13 nm, (Fig. 4E–4F) 3.5 nm Cr-AuNp. Red arrows indicate extracellularly bound Cr-AuNp and green arrows/circle indicate internalized particles. (Fig. 4G–4H) show 3.5 nm and 13 nm probes used in Cr-AuNp preparation.

Mentions: Unlike mammalian cells, bacteria lack the endocytosis mechanisms to uptake nanoparticles or biomolecules [39]. When S. oneidensis cells were incubated with untreated gold nanoparticles (3.5 nm/13 nm) in LB broth (13 nm shown in Figure 4b), the organisms did not internalize particles irrespective of the colloidal concentration and the incubation time. When the particles were functionalized with thiolated-PEG (SH-PEG, MW 5000), an increase in stability of the probes was observed, but there was no noticeable change in their uptake (data not shown). In addition, no uptake of these particles was observed when incubated in the presence of chromate in the incubating matrix (data not shown). However, a drastic improvement in the uptake (internalization) of the particles by cells was noted when Cr-AuNps were used as shown in Figure 4c–f. S. oneidensis MR-1 internalized both 3.5 nm and 13 nm gold nanoparticles when decorated with chromate (K2CrO4). In addition to 3.5 nm and 13 nm nanoprobes, we also attempted uptake experiments with 40 nm sized chromate coated probes. However, the bacterial uptake achieved with 40 nm probes was considerably lower than that attained with the use of 3.5 nm or 13 nm particles. While we have not characterized the transport mechanism responsible for this uptake, it is very likely that the Cr-conjugated gold nanoparticles are taken up through sulfate transport systems. It is accepted that bacterial chromate uptake occurs through sulfate transporters due to the structural similarity of chromate and sulfate ions. Competitive inhibition of sulfate uptake by chromate has also been demonstrated [40], [41]; however, internalization could possibly occur through dedicated chromate receptors that are yet to be characterized. Multiple levels of interaction ranging from mere physical binding to the cell surface to complete internalization of Cr-AuNp probes into the bacterial cells could occur. Since the cells were thoroughly washed prior to imaging, an almost complete removal of non-specifically bound probes is assured.


Surface-enhanced Raman imaging of intracellular bioreduction of chromate in Shewanella oneidensis.

Ravindranath SP, Henne KL, Thompson DK, Irudayaraj J - PLoS ONE (2011)

Thin-section TEM Images of S. oneidensis MR-1.A. without particles, B. plain 13 nm gold Nanoparticles, Fig. 4C–4D. Chromate coated gold nanoparticles, Cr-AuNp:13 nm, (Fig. 4E–4F) 3.5 nm Cr-AuNp. Red arrows indicate extracellularly bound Cr-AuNp and green arrows/circle indicate internalized particles. (Fig. 4G–4H) show 3.5 nm and 13 nm probes used in Cr-AuNp preparation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045368&req=5

pone-0016634-g004: Thin-section TEM Images of S. oneidensis MR-1.A. without particles, B. plain 13 nm gold Nanoparticles, Fig. 4C–4D. Chromate coated gold nanoparticles, Cr-AuNp:13 nm, (Fig. 4E–4F) 3.5 nm Cr-AuNp. Red arrows indicate extracellularly bound Cr-AuNp and green arrows/circle indicate internalized particles. (Fig. 4G–4H) show 3.5 nm and 13 nm probes used in Cr-AuNp preparation.
Mentions: Unlike mammalian cells, bacteria lack the endocytosis mechanisms to uptake nanoparticles or biomolecules [39]. When S. oneidensis cells were incubated with untreated gold nanoparticles (3.5 nm/13 nm) in LB broth (13 nm shown in Figure 4b), the organisms did not internalize particles irrespective of the colloidal concentration and the incubation time. When the particles were functionalized with thiolated-PEG (SH-PEG, MW 5000), an increase in stability of the probes was observed, but there was no noticeable change in their uptake (data not shown). In addition, no uptake of these particles was observed when incubated in the presence of chromate in the incubating matrix (data not shown). However, a drastic improvement in the uptake (internalization) of the particles by cells was noted when Cr-AuNps were used as shown in Figure 4c–f. S. oneidensis MR-1 internalized both 3.5 nm and 13 nm gold nanoparticles when decorated with chromate (K2CrO4). In addition to 3.5 nm and 13 nm nanoprobes, we also attempted uptake experiments with 40 nm sized chromate coated probes. However, the bacterial uptake achieved with 40 nm probes was considerably lower than that attained with the use of 3.5 nm or 13 nm particles. While we have not characterized the transport mechanism responsible for this uptake, it is very likely that the Cr-conjugated gold nanoparticles are taken up through sulfate transport systems. It is accepted that bacterial chromate uptake occurs through sulfate transporters due to the structural similarity of chromate and sulfate ions. Competitive inhibition of sulfate uptake by chromate has also been demonstrated [40], [41]; however, internalization could possibly occur through dedicated chromate receptors that are yet to be characterized. Multiple levels of interaction ranging from mere physical binding to the cell surface to complete internalization of Cr-AuNp probes into the bacterial cells could occur. Since the cells were thoroughly washed prior to imaging, an almost complete removal of non-specifically bound probes is assured.

Bottom Line: Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells.Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported.While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

View Article: PubMed Central - PubMed

Affiliation: Bindley Bioscience Center, Birck Nanotechnology Center, Agricultural and Biological Engineering, Purdue University, West Lafayette, Indiana, United States of America. josephi@purdue.edu

ABSTRACT
This proposed research aims to use novel nanoparticle sensors and spectroscopic tools constituting surface-enhanced Raman spectroscopy (SERS) and Fluorescence Lifetime imaging (FLIM) to study intracellular chemical activities within single bioremediating microorganism. The grand challenge is to develop a mechanistic understanding of chromate reduction and localization by the remediating bacterium Shewanella oneidensis MR-1 by chemical and lifetime imaging. MR-1 has attracted wide interest from the research community because of its potential in reducing multiple chemical and metallic electron acceptors. While several biomolecular approaches to decode microbial reduction mechanisms exist, there is a considerable gap in the availability of sensor platforms to advance research from population-based studies to the single cell level. This study is one of the first attempts to incorporate SERS imaging to address this gap. First, we demonstrate that chromate-decorated nanoparticles can be taken up by cells using TEM and Fluorescence Lifetime imaging to confirm the internalization of gold nanoprobes. Second, we demonstrate the utility of a Raman chemical imaging platform to monitor chromate reduction and localization within single cells. Distinctive differences in Raman signatures of Cr(VI) and Cr(III) enabled their spatial identification within single cells from the Raman images. A comprehensive evaluation of toxicity and cellular interference experiments conducted revealed the inert nature of these probes and that they are non-toxic. Our results strongly suggest the existence of internal reductive machinery and that reduction occurs at specific sites within cells instead of at disperse reductive sites throughout the cell as previously reported. While chromate-decorated gold nanosensors used in this study provide an improved means for the tracking of specific chromate interactions within the cell and on the cell surface, we expect our single cell imaging tools to be extended to monitor the interaction of other toxic metal species.

Show MeSH
Related in: MedlinePlus