Chemically individual armoured bioreporter bacteria used for the in vivo sensing of ultra-trace toxic metal ions

Zhijun Zhangab, Enguo Juab, Wei Bingab, Zhenzhen Wangab, Jinsong Rena and Xiaogang Qu*a
aLaboratory of Chemical Biology and State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, China. E-mail:
bUniversity of Chinese Academy of Sciences, Beijing 100039, China

Received 16th May 2017 , Accepted 20th June 2017

First published on 20th June 2017

A chemically engineered armour is developed for simultaneously improving bioreporter bacterial vitality and shielding infectivity. The armour can help bacteria to resist various insults and even immune phagocytosis. Meanwhile, the bacterial infectivity has proven to be greatly shielded as well. Most importantly, the original bacterial biosensing activity is well preserved, which is competent for sensing trace arsenic in water, serum, and even in vivo.

Recent progress in gene circuit engineering and systems biology has dramatically improved our ability to rationally engineer bacteria with numerous novel biological functions.1 The construction of bioreporter bacteria with exceptional biosensing ability was identified as one of the classic accomplishments in this field.2 Through tailored gene circuits, bioreporter bacteria can dose-dependently translate various chemical inputs into a quantifiable reporter protein output.3 Such genetically engineered bacteria have recently been used in environmental monitoring,4–7 health care,8 food inspection,9 pharmaceutical screening,10 biological research,11–14 etc. However, the usual quota of bacterial weaknesses and non-negligible infectivity brings many restrictions to their practical applications, especially under tough conditions. On the one hand, they are fragile in a harsh environment and can be easily inactivated by various insults, like hydrolytic enzymes, viruses, and toxic nanoparticles. In addition, for in vivo applications, they may be quickly cleared by the host immune system.15 On the other hand, the diverse virulence factors and high proliferation rate make them potentially dangerous to humans, especially to the experimenters. Therefore, improving the self-protection ability and shielding infectivity are of significant importance for the advanced application of bioreporter bacteria.

In nature, some unicellular organisms dress themselves with armour to improve their vitality.16–20 For example, diatom is encapsulated with a sophisticated porous silica shell (frustule) to protect the organism against predators, but facilitate gas, nutrient and metabolite exchange;21 bacteria such as Pseudomonas aeruginosa and Streptococcus pneumonia can produce high molecular weight polysaccharide shells (capsules) to evade host immune clearance;18 the spore released from Bacillus subtilis is encased in a thick protein shell to keep insults away and remain dormant through tough times.19 Inspired by the functions and structures of these natural armours, we consider that the fragility and infectivity of bioreporter bacteria can be conquered by well-designed artificial armour. This armour should be fabricated in gentle conditions, with a porous structure, chemical inertness, optical transparency and high stability. Although recently extensive research efforts have been focused on developing advanced single-cell encapsulation techniques,22–31 achieving an artificial armour to satisfy all these requirements is still a great challenge.

Mesoporous silica (mSi), as a frequently used biocompatible material with chemical inertness, semipermeability, optical transparency and high mechanical strength, would be an ideal candidate to fabricate this demanding artificial armour.32 Consequently, to demonstrate our hypothesis, we first developed a simple and amicable way to uniformly and individually encapsulate the Escherichia coli DH5α strain with a sophisticated mSi shell. The schematic of the encapsulation process is illustrated in Fig. 1a. Briefly, bacteria harvested from an LB culture medium were dispersed in 10.3% saccharose isotonic solution with Pluronic®F-127, (3-aminopropyl) triethoxysilane (APTES) and tetraethoxysilane (TEOS) as the mineralization agents. After agitating at room temperature overnight, a white precipitant of encapsulated bacteria was obtained. This one-pot, toxicity-free and isotonic synthesis would efficiently avoid damage from a complex synthesis procedure,33 extra toxic reagents24,29,30 like acids, alkalis, alcohols or cationic polymers, and also the osmotic pressure in a low salt encapsulation system.24,25 Meanwhile, the biocompatible nonionic surfactant Pluronic®F-127 would help to form a fine and stable mesoporous structure. Fig. 1b–e shows the typical SEM and TEM images of the bacteria before and after encapsulation with mSi shells. It was found that the bare bacteria greatly shriveled during drying. However, after being individually encapsulated the armoured bacteria became more stable and presented a plump rod shape with a smooth surface. The high resolution SEM image (Fig. 1c) reveals the mesoporous structure of the shell, and the diameter of most of the pores was less than 10 nm. The TEM images and corresponding elemental mappings (Fig. 1e and f) further certified that the bacteria were uniformly encapsulated with the mSi shell. The mSi shell (the magenta layer in e) wrapped tightly around the outside cell wall (the green layer in e) and was measured to be about 50 nm in thickness. The N2 absorption–desorption isotherms in Fig. S1 (ESI) also reveal the mesoporous structure of the armour. As a comparison, bacteria that were encased with a silica shell through a conventional method34,35 were also prepared (Fig. S2, ESI).

image file: c7cc03794e-f1.tif
Fig. 1 (a) Schematic representation of the individual encapsulation of bacteria with mesoporous silica. The SEM images of (b) bare DH5α and (c) individually silicified DH5α (DH5α@mSi). The TEM images of (d) bare DH5α and (e) DH5α@mSi (the cell membrane is colored green and mSi layer is colored magenta in the enlarged TEM image). (f) The dark-field TEM image and corresponding TEM elemental mappings of the C, N, O and Si signals of DH5α@mSi.

After being chemically engineered with the mSi armour, the viability of the bacteria (DH5α@mSi) was first evaluated through fluorescein diacetate (FDA)–propidium iodide (PI) double staining tests. As seen in Fig. 2b, above 95% of the armoured bacteria were found to be still alive. The high viability mostly benefits from the amiable chemical engineering process. Contrarily, the conventional method encased bacteria (DH5α@Si) exhibited a low viability of 76% (Fig. 2c).

image file: c7cc03794e-f2.tif
Fig. 2 The fluorescence images of FDA-PI double stained (a) DH5α, (b) DH5α@mSi and (c) DH5α@Si, and the insets are pie charts of the survival ratio (the live bacteria are in green, and the dead bacteria are in red). Flow cytometry illustrates the activity of bare 1598 (d) and 1598@mSi (e) to different concentrations of sodium arsenite.

The impact of chemical engineering on the bacterial activity was then investigated. In view of the importance of arsenic detection,36,37 here an arsenic responsive Escherichia coli DH5α bioreporter strain 1598 was chosen as a model, which was engineered by the group of Professor Jan Roelof van der Meer.38 The working principle of 1598 is outlined in Fig. S3 (ESI), in which the expression of the enhanced green fluorescent protein (egfp) is designed to be activated only when arsenic appears. All the bacterial biosensors based on this strain have been demonstrated to be highly sensitive.8,38 Intriguingly, as presented in Fig. 2d and e and Fig. S4 (ESI), after being dressed with the mSi shell, the bacterial arsenic responsive activity was maintained with some attenuation. The activity attenuation of encapsulated bacteria is considered to be unavoidable39 which is mostly because of the decrease in the metabolic activity caused by proliferation restriction (Fig. S5, ESI). As a comparison, the activity of the conventional method encapsulated bioreporter bacteria (1598@Si) was also investigated. It was found that 1598@Si couldn’t produce any detectable response signal even under a high arsenic concentration of 500 μg L−1 (Fig. S4, ESI). We consider that, except for the limited nutrition uptake (Fig. S6, ESI) and proliferation (Fig. S5, ESI) ability, the probable blocking of the arsenic uptake ion channel by the silica shell would also contribute to an inhibited biosensing activity of 1598@Si.40

We next investigated whether this artificial armour had the same functions as those of the natural armours. Self-protection is the most important function of the armours of natural unicellular organisms. They only permit beneficial molecules like oxygen and nutrients to transport across and sustain life, but they keep dangers like hydrolases, viruses and toxic nanoparticles away to protect the organisms. Here, we first studied the selective permeability of our fabricated armour. The results in Fig. S6 and S7 (ESI) reveal that the artificial armour can facilitate small molecule (4′,6-diamidino-2-phenylindole (DAPI) as a model) fast transport but strongly resists the model nanoparticles (Fig. S8a, ESI). Then the threat resistance capability towards two representative threats, lysozyme (Lys) and AgNPs (Fig. S8b, ESI), was further investigated. As shown in Fig. 3a, the bare bacteria were very fragile to Lys and AgNPs, with over 95% of them being quickly killed, however over 90% of the armoured bacteria were still alive.

image file: c7cc03794e-f3.tif
Fig. 3 (a) The FDA-PI double staining fluorescence images of DH5α and DH5α@mSi after being invaded by Lys and AgNPs. The flow cytometry results of the phagocytosis of 1598egfp (b) and 1598egfp@mSi (c) by RAW264.7 at different ratios. (d) The confocal images of the phagocytosis of 1598egfp and 1598egfp@mSi (green rod) by RAW264.7 (the membranes were stained in red, the nuclei were stained in blue) at a bacteria to RAW264.7 ratio of 100[thin space (1/6-em)]:[thin space (1/6-em)]1.

Helping the organism to evade the host immune clearance by shielding the surface antigenic targets is another important self-protection manner of some natural armours.41 For the armoured bacteria, the bacterial surface was strictly hidden. Thus, we considered that they possessed the ability to evade immune phagocytosis as well. To certify this point, phagocytosis by a model phagocyte murine macrophage cell line RAW264.7 was performed. The flow cytometry results in Fig. 3b and c reveal that bare 1598egfp (egfp expressed 1598) can be efficiently phagocytosized, and the amount of phagocytosis increased with increasing the bacteria to RAW264.7 ratio. However, much less 1598egfp@mSi could be phagocytosized under the same conditions. Similar results were also observed from the cell images in Fig. 3d, which shows the typical confocal fluorescence images of the bacteria phagocytosized by RAW264.7 at a ratio of 100[thin space (1/6-em)]:[thin space (1/6-em)]1. Dozens of bare 1598egfp were found to be phagocytosized by a single RAW264.7 cell, whereas in contrast, only a few 1598egfp@mSi could be phagocytosized. All these results demonstrate that the mSi armour could help the bacteria to minimize host immune phagocytosis.

After demonstration of the natural armour like defensibility, the virulence shielding capacity of our designed artificial armour was then studied. The cytotoxicity of bare 1598 and 1598@mSi at the cellular level was first investigated using RAW264.7 (immunocyte), 3T3 (bistiocyte) and erythrocyte as model cells. As shown in Fig. 4a and b and Fig. S9 (ESI), although 1598 is not a pathogenic strain, it presented considerable cytotoxicity, especially towards the RAW264.7 and 3T3 cells. Interestingly, 1598@mSi displayed a tolerable cytotoxicity even at a high bacterial concentration. Then the infectivity of bare 1598 and 1598@mSi were tested in a mouse skin infection model. As illustrated in Fig. 4c, bare 1598 caused an obvious inflammatory response in mice. The skin of the injected area became red and swollen, and this symptom was found to last at least 15 days with a tendency to worsen. However, armoured 1598 didn’t induce any changes in the skin. In addition, the body weight and organs of the mice were also not influenced by 1598@mSi (Fig. S10 and S11, ESI). These results indicate that the virulence of the bacteria could be greatly inhibited through surface chemical engineering.

image file: c7cc03794e-f4.tif
Fig. 4 The viability of (a) the RAW264.7 cells and (b) the 3T3 cells incubated with different concentrations of bacteria. (c) The changes in the skin (the red circles point out the injection sites) of mice up to 15 days after being subcutaneously injected with 50 μL 1598 and 1598@mSi.

Up to now, we have demonstrated the simultaneous self-protection and infectivity shielding capacity of armoured bioreporter bacteria, and consequently the serviceability was finally investigated. As presented in Fig. 5a, arsenic detection in water was found to be highly sensitive with a detection limit of 7.1 μg L−1. Notably, although this value is higher than 2.5 μg L−1 of bare 1598 (Fig. S12, ESI), it is still meaningful for practical applications since 10 μg L−1 is the maximum allowable level of arsenic in drinking water according to the guidelines from the U.S. Environmental Protection Agency (EPA) and World Health Organization (WHO).42,43 Then the detection in more complex samples was investigated. Fig. 5b illustrates that, even in serum, spiked arsenic can be sensitively detected with a very low detection limit of 3.4 μg L−1. After that, the in vivo sensing ability of this armoured bioreporter bacteria was evaluated on an arsenic exposed mouse model. As presented in Fig. 5c, the arsenic exposed mice at the 1/20 LD50 level were able to generate a detectable fluorescence signal. These results indicate that the armoured bioreporter bacteria are qualified for sensitive in vitro and even in vivo sensing.

image file: c7cc03794e-f5.tif
Fig. 5 The fluorescence spectra of arsenic detection in water (a) and serum (b) using 1598@mSi as probes, λex = 490 nm. The concentrations of spiked sodium arsenite were 0, 10, 25, 50, 100, 250, 500 and 1000 μg L−1. (c) The fluorescence imaging results of in vitro and in vivo (within the white circles) arsenic sensing. The mice, from left to right, are: the one injected with 0.9% NaCl and the sodium arsenite exposed mice with final arsenic concentrations of 336 (1/50 LD50), 840 (1/20 LD50) and 3360 (1/5 LD50) μg kg−1, respectively.

In summary, a simple one-pot and amiable single-cell encapsulation method was developed for individually armoured live bioreporter bacteria with a sophisticated mesoporous silica shell. This artificial armour not only greatly improved the bacterial vitality in a harsh environment and even the ability to minimize immune phagocytosis, but also deeply shielded the bacterial infectivity both in vitro and in vivo. Meanwhile, the native arsenic biosensing activity of the model bioreporter bacterium was well preserved. Using this armoured bioreporter strain, trace amounts of arsenic in water, serum and even in vivo could be quantified. We believe that this powerful chemical method is qualified for armouring other functionalized bacteria for advanced applications.

The authors thank Professor Jan Roelof van der Meer for kindly sharing the Escherichia coli DH5α bioreporter strain 1598. The financial support was provided by the 973 project (2012CB720602) and NSFC (21210002, 21431007, 21533008).


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Electronic supplementary information (ESI) available: Experimental section and additional results. See DOI: 10.1039/c7cc03794e

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