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Rapid isolation of #-glutamyl-transferase rich-bacteria from mouse gut by a near-infrared fluorescent probe with large Stokes shift Tao Liu, Qiulong Yan, Lei Feng, Xiaochi Ma, Xiangge Tian, Zhenlong Yu, Jing Ning, Xiaokui Huo, Cheng-Peng Sun, Chao Wang, and Jing-Nan Cui Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02118 • Publication Date (Web): 20 Jul 2018 Downloaded from http://pubs.acs.org on July 21, 2018

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Analytical Chemistry

Rapid isolation of γ-glutamyl-transferase rich-bacteria from mouse gut by a near-infrared fluorescent probe with large Stokes shift Tao Liu,†,§ Qiu-Long Yan,‡,§ Lei Feng,†,‡,#,§ Xiao-Chi Ma,‡ Xiang-Ge Tian,‡ Zhen-Long Yu,‡ Jing Ning,‡ Xiao-Kui Huo,‡ Cheng-Peng Sun,‡ Chao Wang,‡,* Jing-Nan Cui†,* † State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China. Corresponding authors: Chao Wang, Jing-Nan Cui (E-mail: [email protected], [email protected]) ‡ College of Pharmacy, Academy of Integrative Medicine, Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China # Institute of Functional Materials and Molecular Imaging, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China ABSTRACT: Bacterial γ-Glutamyltranspeptidases (γ-GT) is a well-known metabolic enzyme, which could cleave the γ-glutamyl amide bond of γ-glutamyl analogues. As a key metabolic enzyme of bacteria and a virulence factor for the host, bacterial γ-GT was determined to be a novel pharmaceutical target for new anti-biotics development. However, there is no efficient method for the sensing of γ-GT activity in bacteria and the recognition of γ-glutamyltransferase rich-bacteria. In the present work, a dicyanoisophorone derivative (ADMG) has been designed and developed to be a sensitive and selective near-infrared fluorescent probe for the sensing of bacterial γ-GT. ADMG not only sensed bacterial γ-GT in vitro, but also imaged intestinal bacteria in vivo. More interesting, the intestinal bacteria existed in the duodenum section of mouse displayed significant fluorescence emission. Under the guidance of the sensing of γ-GT using ADMG, three intestinal bacteria strains K. pneumoniae CAV1042, K. pneumoniae XJRML-1, and E. faecalis were isolated successfully, which expressed the bacterial γ-GT. Therefore, the fluorescent probe ADMG not only sensed the endogenous bacterial γ-GT and imaged the intestinal bacteria, but also guided the isolation of intestinal bacteria possessing γ-GT efficiently, which suggested a novel biological tool for the rapid isolation of special bacteria from mixed sample.

γ-Glutamyltranspeptidase (γ-GT; EC 2.3.2.2) is an ubiquitous heterodimeric metabolic enzyme, which was determined to exist in bacteria, such as Escherichia coli, Campylobacter jejuni, Francisella tularensis, Helicobacter bilis, Helicobacter pylori, Thermus thermophiles, Deinococcus radiodurans, and Bacillus subtilis.1-3 Bacterial γ-GTs from different bacteria exhibit high similarities in their primary structures, which were extracellular or localized in the periplasmic space of bacteria.1,4 Generally, γ-GT could cleave the γ-glutamyl amide bond of γ-glutamyl derivatives, such as glutathione, as well as transfer the γ-glutamyl to water, or amino acid, even peptide.1 The S-conjugates of glutathione also could be degenerated by γ-GT on the basis of the cleavage of γ-glutamyl amide bond. In bacteria, γ-GT generally could catalyze the initial step of the degradation of the extracellular GSH into its amino acids constituents, which are transported into the cell and reused in the protein biosynthesis. Thus, γ-GT was a key enzyme for the metabolism of bacteria, an important virulence factor for colonization and bacterial immune evasion, which was recognized to be a novel pharmaceutical target for the antibiotics development. On the other hand, some evidences indicated that bacterial γ-GT also played an important role as virulence factor in bone destruction of host.4 Additionally, γ-GT also mediated the depletion of extracellular glutathione, which led to oxidative stress for surrounding cells of host. Furthermore, γ-GT from Helicobacter bilis was determined to be a virulence factor decreasing host cells viability,2,5 and could be

responsible for induction of inflammatory disorders of host cells. As a biocatalyst, bacterial γ-GT was considered as a useful tool to produce γ-glutamyl derivatives, more soluble and stable with respect to the non-modified precursors or to be employed as glutaminase in food industry.1 Thus, bacterial γ-GTs displayed important role for many industrial applications, such as the biosynthesis of various γ-glutamyl derivatives, theanine, flavor enhancers, medical substances, and the glutaminase application in the food industry.6 Above all, bacterial γ-GT was recognized as a virulence factor for the host, and a novel target for the anti-biotics. So, the wide applications of bacterial γ-GT in biotechnological, pharmaceutical as well as in food industry desired a rapid, sensitive, and selective detection method. On the other hand, rapid identification of these bacteria expressed γ-GT would be helpful for the anti-biotics development and the γ-GT isolation, even the treatment of disease induced by bacterial γ-GT. Compared with the bacterial γ-GT, γ-GT from mammals have been found the roles in the cell defense against oxidative stress, Parkinson's disease, other neurodegenerative diseases, and cardiovascular disease.7 In particular, γ-GT attracted plenty of attention as a tumor-related enzyme, and a higher level of γ-GT in serum is reported as a risk factor of cancer. Therefore, various γ-GT activated fluorescent probes have been developed to detect mammalian γ-GT in vitro and in vivo.8-24 However, there is no NIR fluorescent probe for sensing of γGT activity in bacteria, and further isolation of γ-glutamyl-

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transferase rich-bacteria from intestinal flora. It was known that NIR fluorescence probes with a remarkable Stokes shift were widely applied for imaging biomolecules in vivo,25-27 such as (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2enylidene) malononitrile (ADM), a dicyanoisophorone derivative.28-35 In the present work, a conjugated derivative based on glutamic acid and ADM was synthesized as an ADMglutamate (ADMG), which was developed as a NIR fluorescent probe displayed high sensitivity and selectivity towards γGT (Scheme 1), which displayed the advantages for the little interferences of autofluorescence of biomaterial and strong penetrability. The γ-glutamyl amide bond not only was designed as the γ-GT-triggered target but also improved the water solubility of the NIR fluorescent probe ADMG. Using ADMG, the bacterial γ-GT was sensed and imaged for intestinal bacteria. Furthermore, with the guidance the ADMG, the intestinal bacteria expressed γ-GT were isolated and identified from the mouse efficiently.

Scheme 1. Illustration for the degeneration of ADMG mediated by bacterial γ-GT and its fluorescence response towards γ-GT. EXPERIMENTAL SECTION Materials and instruments. All of the reagents were purchased from J&K Scientific. γ-Glutamyltranspeptidases (γGT), Proteinase K, Carboxylesterases (CES2), Human albumin (HA), Dipeptidyl peptidase 4 (DPP 4), Leucine arylamidase (LAP), Prolidase (PAP), Trypsin, Pepsin, and Lipase were obtained from Sigma-Aldrich. The fluorescence intensities were measured on BioTek Synergy H1 microplate reader (BioTek, USA). NMR spectra were acquired by a Bruker 501. Constant temperature incubator shaker (ZHWY-2012C) was the production of Shanghai Zhicheng Analytical Instrument Co. Ltd (P.R. China). The confocal fluorescence images of bacteria were required using Olympus FV 1000 MPE/FV-1000 FLIM Confocal Microscope with the excited wavelength λex 488 nm and emission filter of λem 620 – 680 nm (Olympus, Japan). The imaging of mice and gut were recorded in a NightOWL II LB983 small animal in vivo imaging system equipped with a sensitive Charge Coupled Device (CCD) camera, with an excitation laser of 480 nm and an emission filter of 650 – 670 nm. Synthesis and characterization of ADMG. The fluorescent probe ADMG was synthesized on the basis of the fluorescent fluorophore ADM (Scheme S1), which was synthesized according to the literature.35 The structures of ADMG and ADM were determined using 1H NMR, 13C NMR and HR-MS. (Figure S1-S9) Activity assay of bacterial γ-GT using ADMG. Bacterial γ-GT (0.72 U/mL) was dissolved in KH2PO4-K2HPO4 buffer (pH 8.0). Then, the fluorescent probe ADMG (10 µM) was

added into the enzyme solutions for co-incubation at 37 °C for 10 min. The enzymatic reaction was stopped using acetonitrile (33%, v/v) and acquired the fluorescence intensity using microplate reader (λex = 456 nm). Selectivity study of ADMG towards γ-GT. In order to confirm the selectivity of ADMG for bacterial γ-GT, ADMG was incubated with various enzymes such as Proteinase K, Carboxylesterases (CES2), Human Albumin (HA), Dipeptidyl peptidase 4 (DPP 4), Leucine arylamidase (LAP), Prolidase (PAP), Trypsin, Pepsin, and Lipase in the standard incubation system at 37 °C for 30 min with the concentration of 0.1 mg/mL, respectively. Furthermore, the interferences of other species, including amino acids (cysteine, glycine, tryptophan, tyrosine, L-arginine, GSH), metal ions (Ca2+, K+, Na+, Mg2+, Fe3+, Sn4+), oxidants (MnO2) and reducers (Vitamin C, NaB H4, Na2S2O3) were performed for the fluorescence response of ADMG towards bacterial γ-GT. Fluorescence imaging of intestinal bacteria using ADMG. Different bacterial strains were cultured on LuriaBertani (LB) liquid medium for 24 h at 37 °C. When the culture of bacteria suspension were diluted to 1.0 × 109 cells/mL, ADMG (50 µM) was added for an incubation of 1 h. After the clean out of the medium, the bacterial cells were suspended in PBS solution, which were dropped on slide glasses for imaging experiment. The bacteria were imaged using Confocal Microscope with λex 488/λem 620 – 680 nm. To study the enzyme specificity, the bacteria were pretreated with enzyme inhibitor acivicin (ACI, 5 – 100 µM) and labeled with ADMG for imaging studies. In vivo imaging of intestinal bacteria in Mouse. Mice (male, 20 g) were housed under specific pathogen-free conditions and received food and water ad libitum. Depletion of gut microbiota was achieved by administering mice broadspectrum antibiotics. The antibiotic regimen mainly included ampicillin, metronidazole, neomycin and vancomycin. Ampicillin was added into the tap drinking water at a final concentration of 1 g/L. Metronidazole (100 mg/kg), neomycin (100 mg/kg) and vancomycin (50 mg/kg) were oral administrated by gavage twice a day. Metronidazole, neomycin and vancomycin were oral administrated by gavage twice a day at concentration of 1 and 0.5 g/L, respectively. Mice in control group received an equivalent volume of physiological saline every day. After a week, mice were anesthetized and then intragastric administration with ADMG (100 µM) for imaging in vivo with λex 480/λem 650 – 670 nm. Isolation and identification of intestinal bacteria. In the intestinal distribution of γ-GT experiment, mice fasted for 24 h (free access to water) were anesthetized, and the whole intestinal tracts of which were obtained and divided into six segments: 2 duodenum; 3 − 6 small intestine (3 − 4 jejunum, upper small intestine; 5 and 6 ileum, lower small intestine) and 7 colon. The stomach was assigned as 1. Each intestinal segment was flushed with cold saline and immediately placed in 37 °C phosphate buffer solution. The reaction was started by adding ADMG (100 µM). Following 30-min incubation, the segments were washed by phosphate buffer three times and then imaged by the small animal imaging system, with excitation at 480 nm and semiconductor laser emission collected at 650 – 670 nm. For the intestinal segment with significant fluorescence emission from three individual mice, the corresponding intestinal contents were collected and suspended in the saline.


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Analytical Chemistry Then, the suspension solutions were coated on the LB-Agar plates at 37 °C for 24 h. When the bacterial colonies were observed, ADMG (100 µM) was added to the colonies for a co-incubation of 1 h at 37 °C. The fluorescence images of bacterial colonies were recorded using small animal imaging system (λex 480/λem 650 – 670 nm). These bacterial colonies displayed significant fluorescence emission would be collected and identified using 16sDNA analysis. RESULTS AND DISCUSSION Designation and fluorescence characteristics of ADMG for the detection of γ-GT. It was known that the bacterial γGT could cleave the γ-glutamyl amide bond of γ-glutamyl derivative, which could be applied for the anti-biotics development, and industrial enzyme. On the basis of the special function of γ-GT, a NIR fluorescent probe (ADMG) derived from the adduct of (E)-2-(3-(4-aminostyryl)-5,5dimethylcyclohex-2-enylidene)malononitrile (ADM) and glutamic acid was designed and developed for the detection of bacterial γ-GT. The γ-glutamyl amide bond of ADMG was preferred to be cleaved by bacterial γ-GT, yielded the productions of glutamic acid and ADM fluorophore (Scheme 1). Under the excitation at λex 456 nm, a significant fluorescence emission at λem 670 nm was observed for ADM, compared with the no fluorescence emission of ADMG (Figure 1a, 1b). The transformation of ADMG mediated by γ-GT to yield ADM was also confirmed by HPLC-DAD analysis as shown in Figure S10. It was obvious that ADM not only displayed the NIR fluorescence emission, but also possessed the large Strokes shift prosperity. Because the introduction of the glutamic acid moiety, ADMG displayed good water solubility as 300 µM (DMSO < 1%, v/v), which was suitable for the application in the enzymatic system, even the culture of bacteria. Compared with ADMG, the sensing product ADM displayed stronger fluorescence emission at λem 670 nm (Ф = 0.08, pKa (2.18). The cytotoxicities of ADMG and ADM were also investigated using human cancer cells A549 and HepG2. As shown in Figure S11, ADMG displayed no cytotoxicity against A549 and HepG2 at 50 µM. ADM displayed weak

Figure 1. (a) Absorption spectra and (b) emission spectra of ADMG (10 µM) and its metabolite ADM (10 µM). (c) Fluorescence spectra changes of ADMG (10 µM) upon addition of increasing concentrations of γ-GT (0 ‒ 1.4 U/mL) with the 10 min incubation time. (d) Fluorescence spectra changes of ADMG (10 µM) upon addition of γ-GT (0.72 U/mL) for 0 ‒ 20 min incubation. (Enzymatic reaction system: KH2PO4K2HPO4 buffer, pH 8.0, 37 °C; Measurement system: phosphate buffer-acetonitrile v/v = 2:1, λex = 456 nm)

cytotoxicity against A549 and HepG2 at 50 µM. Therefore, ADMG was developed to be the NIR fluorescent probe for the detection of bacterial γ-GT, with the γ-glutamyl amide bond designed as the trigger. As a NIR fluorescent probe, the fluorescence intensity at 670 nm towards different concentrations of γ-GT displayed a good linearity at the range 0 – 1.4 U/mL (Figure 1c, and Figure S12) and the limit of detection (3σ/slope) was estimated as 0.036 U/mL, which indicated the good affinity and the sensitive detection of ADMG towards bacterial γ-GT. On the other hand, the fluorescence behavior of ADMG towards bacterial γ-GT also displayed the time-dependent characteristic at the incubation time from 0 to 20 min, which also suggested the fast reaction velocity for the hydrolysis of ADMG mediated by bacterial γ-GT (Figure 1d, S13). The interference on the fluorescence intensities of ADM and ADMG of the incubation pH values were investigated, which indicated the good stability of ADMG and ADM with the pH ranged from 3 to 12 (Figure S14). Additionally, the optimal pH incubation system was determined as 8.0 for the detection of bacterial γ-GT by ADMG, which was also suitable for the bacteria physiological condition (Figure S14). Meantime, the fluorescence intensities of ADMG towards bacterial γ-GT in various incubated temperature were studied, the results showed that the optimal incubated temperature for the bacterial γ-GT activity was 37 °C (Figure S15). Thus, ADMG was established for the detection and activity assay of bacterial γ-GT in vitro, with the characteristics of large Stokes shift. Selectivity of ADMG towards bacterial γ-GT. In consideration of the application the fluorescent probe ADMG for the sensing of endogenous bacterial γ-GT in bacteria cells, even for the intestinal bacteria in vivo, the selectivity of ADMG towards bacterial γ-GT should be studied for other biological enzymes. In the present work, various enzymes and proteins possessing the hydrolase functions including proteinase K, carboxylesterases (CES2), human albumin (HA), dipeptidyl peptidase 4 (DPP 4), leucine arylamidase (LAP), prolidase (PAP), trypsin, pepsin, and lipase were applied to hydrolysis the probe ADMG. Compared with the fluorescent intensity of ADMG towards bacterial γ-GT, no fluorescence emission was observed for the other enzymes groups (Figure 2a), which suggested the special property of ADMG towards bacterial γGT. Furthermore, no interference of other species including amino acids, metal ions, oxidants and reducers was observed for bacterial γ-GT assay using ADMG (Figure 2b). Thus,

Figure 2. (a) Fluorescence intensity of ADMG (10 µM) upon addition of γ-GT (0.1 mg/mL) and various hydrolases (0.1 mg/mL) respectively (30 min incubation). (b) Fluorescence responses of ADMG (10 µM) to various interfering species, including γ-GT (0.72 U/mL), metal irons (2 mM), amino acids (1 mM). (λex = 456 nm, λem = 670 nm)



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ADMG could be applied as the sensitive and selective NIR fluorescent probe for the activity assay and sensing of bacterial γ-GT in vitro and in vitro. Sensing bacterial γ-GT and imaging bacteria using ADMG. In the present work, the ability of ADMG for the sensing of bacterial γ-GT and imaging of bacteria was investigated. After a preliminary screening about the bacteria of our lab, Klebsiella Pneumoniae 1495 was used for the fluorescence imaging experiment. Additionally, acivicin (ACI) is an antineoplastic antibiotic, which was determined to inhibit the enzymatic function of bacterial γ-GT and could be used as the inhibitor of bacterial γ-GT for the imaging experiment.3,36 The bacterium K. Pneumoniae 1495 was cultured in LB medium to afford enough bacterial cells (109 cells/mL). Then, the fluorescent probe ADMG (50 µM) was added into the culture and co-incubated for 1 h. The blank group without ADMG and inhibitory group in the presence of ACI (100 µM) were established at the same time. After the cleanout of culture medium, the bacteria were located at the slide glasses for confocal fluorescence images acquisition. When excited at λex 488 nm, K. Pneumoniae 1495 incubated with ADMG displayed strong fluorescence emission and no fluorescence emission was observed for the blank group (Figure 3a). Meantime, ACI (100 µM) showed significant inhibitory effect on the activity of bacterial γ-GT towards the hydrolysis of ADMG as well as little fluorescence emission observed. Additionally, when K. Pneumoniae 1495 was located at the plate culture medium, ADMG was added to the bacteria colonies for one hour incubation. Then, the bacteria plate was subjected to the animal imaging system with the excited wavelength

λex 480 nm, and strong fluorescence emission at λem 650-670 nm was observed for the bacteria colonies (Figure 3b). So, on the basis of the sensing of bacterial γ-GT, ADMG was applied to image the K. Pneumoniae 1495 cells and colonies successfully. Using the HPLC-DAD, the K. Pneumoniae 1495 culture incubated with ADMG was analyzed for the production of ADM, which also confirmed the application of ADMG to sense γ-GT in bacterial cells (Figure S16). Additionally, a serial fluorescence images have been obtained with the continued laser excitation from 5 – 40 min, which indicated good photostability of ADM as the sensing product (Figure S17). Furthermore, flow cytometric analysis was also performed to confirm the capability of ADMG for the sensing of endogenous γ-GT in K. Pneumoniae 1495 using the passageway FL3 (λex 488/λem > 670 nm) (Figure 3c), which corresponded to the bacterial imaging experiment. When K. Pneumoniae 1495 incubated with ADMG in 96-well plate in the presence of different concentrations of ACI (0 – 100 µM), different fluorescence intensities were observed using animal imaging system. As shown in Figure 3d, this imaging result also indicated the concentration dependent inhibition of ACI against the bacterial γ-GT activity (IC50 6.5 µM). Above all, ADMG was successfully applied to sense bacterial γ-GT and image K. Pneumoniae 1495 using various instruments. Furthermore, ADMG also could be applied for the rapidly screening of inhibitor of endogenous bacterial γ-GT using the 96-well plate. Imaging of intestinal bacteria in vivo using ADMG. Encouraged by the above results, ADMG was used to guide the identification and isolation of intestinal bacteria expressed γGT rapidly. As mentioned above, ADMG has been applied to

Figure 3. (a) Confocal fluorescence images of bacterium Klebsiella Pneumoniae 1495 incubated with ADMG (50 µM, λex 488/λem 620 – 680 nm, Scale bar 25 µm) Fluorescence emission windows: Blank (bacterium with the absence of ADMG), ADMG (bacterium incubated with ADMG), ADMG+ACI (bacterium incubated with ADMG and ACI 100 µM). (b) Fluorescent image of the bacterial plate (K. Pneumoniae 1495 incubated with ADMG 50 µM, λex 480/λem 650 – 670 nm). (c) Flow cytometric analysis of K. Pneumoniae 1495 acquired with the FL3 (λex 488/λem > 670 nm). (d) Concentration dependent inhibition of ACI (0, 5, 10, 30, 50, 100 µM) against the fluorescence intensity of ADMG incubated with K. Pneumoniae 1495.

Figure 4. (a) Fluorescence images of mice (antibiotics+ADMG: mixed antibiotics pre-administrated mouse with intragastric administration of ADMG 100 µM, ADMG: mouse intragastric administrated with ADMG 100 µM, ADMG+ACI: mouse intragastric administrated with ADMG and ACI 100 µM). (b) Fluorescence images of the stomach and intestine from the mouse administrated with ADMG. (c) Fluorescence images of stomach and intestinal segments incubated with ADMG (100 µM). (1) stomach; (2) duodenum; (3 – 6) small intestine (3 − 4 jejunum, upper small intestine; (5) and (6) ileum, lower small intestine) and (7) colon. (λex 480/λem 650 – 670 nm)


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Analytical Chemistry

Figure 5. Fluorescence image (a) and the bright image (b) of intestinal segments incubated with ADMG (100 µM). (2 duodenum; 3 – 6 small intestine: 3 − 4 jejunum, upper small intestine; 5 and 6 ileum, lower small intestine, and 7 colon) (c) Fluorescence image of the mixed bacterial plate from the mouse duodenum (2). (λex 480/λem 650 – 670 nm). image K. Pneumoniae 1495 on the basis of the γ-GT activity successfully. Thus, the capability of ADMG to image intestinal bacteria in the mouse was further investigated. Herein, mice were assigned into four groups, including the blank group with no ADMG administrated, antibiotics group with the intestinal bacteria inhibited by multiple anti-biotics (ampicillin, metronidazole, neomycin and vancomycin) previously before ADMG administrated, ADMG group with the probe ADMG administrated (100 µM), and ADMG + ACI group with ADMG and γ-GT inhibitor ACI administrated. Under the animal imaging system, remarkable fluorescence emission was observed for the mouse just administrated with ADMG (Imax 6.0×e4, Figure 4a, S18) in comparison with no fluorescence emission of the blank group. When the intestinal bacteria were inhibited by multiple anti-biotics, no fluorescence emission displayed for the mouse administrated with ADMG. Similarly, ACI also inhibited the bacterial γ-GT activity significantly as well as no fluorescence emission displayed. These results confirmed that ADMG could detect the bacterial γGT from intestinal bacteria of mouse. For the mouse displayed significant fluorescence emission in the presence of ADMG, the gut was dissected wholly for imaging experiment. As shown in Figure 4b, strong fluorescence emission (Imax 6.6×e4) was observed from the duodenum section in physiology. Thus, the obvious regional property about the fluorescence emission from the mouse gut was interesting, which indicated that the bacteria existed in the duodenum expressed γ-GT highly. In order to confirm this regional property, another blank gut was dissected from a blank mouse and segmented into six sections together with the stomach physiologically. These sections were incubated with ADMG (100 µM) for 30 min and acquired the images excited at 480 nm. As a result, remarkable fluorescence emission (Imax 7.0×e4) was observed for the duodenum section (2), which corresponded to the in vivo imaging experiment. Therefore, ADMG was not only applied to image the intestinal bacteria in vivo successfully, but also suggested the higher expression of γ-GT for these bacteria in duodenum. Isolation of bacteria with high expressed γ-GT under the guidance of ADMG. On the basis of the bacteria imaging experiment in vivo, ADMG could detect endogenous γ-GT of bacteria, which would be helpful for the recognition of γ-GT expressed bacteria from the mixed intestinal bacteria. Under the aseptic condition, the guts of mice were dissected and segmented into six sections, respectively, which were then

incubated with ADMG in 6-well plate for 30 min. Using the animal imaging system, the gut sections were imaged under the excitation λex 480 nm. As a result, the section corresponded to the physiological duodenum (2) displayed significant fluorescence emission (Imax 8.0×e4, Figure 5a, 5b). This experiment was repeated three times with similar results (Figure S19). Then, the intestinal contents of duodenum were collected and diluted by PBS solution. After the centrifugation, the liquid supernatant was coated on the LB culture medium in plate for the bacterial reproduction. When the bacteria colonies were obtained, the plate was divided into blank section and ADMG section with dropwise ADMG was cautiously added to the colonies for 30 min incubation. Under the excitation at 480 nm, seven bacterial colonies (G-1-G-7) displayed strong fluorescence emission (Figure 5c, S20), which suggested the expression of γ-GT. Then, bacterial colonies G-1 – G-7 were cultured in LB medium and identified using 16S DNA analysis experiment. Finally, G-1, G-2, G-3 and G-5 were identified to be K. pneumoniae CAV1042, and G-4, G-7 were identified to be K. pneumoniae XJRML-1, together with E. faecalis (G-6). Thus, the fluorescence sensing of γ-GT using the fluorescent probe ADMG could be applied to recognize those bacteria with γ-GT expression efficiently, which would be a potential bacterial isolation strategy in future. Fluorescence imaging of isolated intestinal bacteria. Under the guidance of the sensing of γ-GT using ADMG, three intestinal bacteria were isolated from the gut of mouse, which were identified to be K. pneumoniae CAV1042, K. pneumoniae XJRML-1, and E. faecalis, respectively. Herein, using the fluorescent probe ADMG, the activities of γ-GT for isolated bacteria were assayed as well as the fluorescence images of isolated bacteria were acquired. Compared with the blank groups, remarkable fluorescence emissions were observed for K. pneumoniae CAV1042, K. pneumoniae XJRML-1, E. faecalis, respectively (Figure 6a, S21, S22, S23). And, E. faecalis displayed weaker fluorescence emission than K. pneumoniae CAV1042 and K. pneumoniae XJRML-1, which could suggest the different activity and expression of γ-GT in bacteria. On the other hand, when the γ-GT inhibitor ACI and ADMG were co-incubated for these isolated bacteria, weak fluorescence emission was observed, which also confirmed the existence of bacterial γ-GT and the capability of ADMG to detect γ-GT activity. Furthermore, flow cytometric analysis has been performed to confirm the fluorescence sensing of γ-GT in three isolated bacteria by ADMG. As shown in Figure 6b, 6c, 6d, significant differences were observed for bacteria incubated with no ADMG, ADMG, and ADMG + ACI, respectively, which were acquired at λex 488/λem > 670 nm. Thus, the fluorescence emission of bacteria deduced from the enzymatic reaction of ADMG mediated by γ-GT was reliable, which could be a novel bacterial isolation strategy for these γ-GT rich-bacteria from mixed sample. CONCLUSIONS In summary, a NIR fluorescent probe ADMG derived from dicyanoisophorone possessing the large Stokes shift was developed for activity assay of bacterial γ-GT sensitively and selectively. And, on the basis of the sensing of endogenous γGT by ADMG, bacterium and mouse have been imaged successfully. The establishment of the assay system about bacterial γ-GT activity would be helpful for the widely application of γ-GT and the discovery of γ-GT inhibitors. Furthermore, the fluorescence imaging of intestinal bacteria on the basis of



Figure 6. Confocal fluorescence images of the isolated bacteria incubated with ADMG (50 µM, λex 488/λem 620 – 680 nm, Scale bar 25 µm). (b, c, d) Flow cytometric analysis of the isolated bacteria K. pneumoniae CAV1042, K. pneumoniae XJRML-1, E. faecalis acquired with the FL3 (λex 488/λem > 670 nm). Blank: bacterium with the absence of ADMG, ADMG: bacterium incubated with ADMG, ADMG+ACI: bacterium incubated with ADMG and ACI 100 µM. γ-GT activity has been applied to guide the recognition and isolation of three intestinal bacterial strains with the expression of γ-GT from mouse intestinal bacteria efficiently, which could be a novel biological technique for the special bacteria isolation. The rapid recognition of γ-GT-rich intestinal bacterial would be helpful for the treatment of these diseases induced by bacterial γ-GT and the corresponding anti-biotics development.

ASSOCIATED CONTENT Supporting Information Supplementary figures and tables; experimental procedures. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected]

Author Contributions §

T. Liu, Q.L. Yan, and L. Feng contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The present work was financially supported by National Natural Science Foundation of China (No. 21572029, 81622047, 81673579, and 81503201), Distinguished professors of Liaoning Provinces, and State Key Laboratory of Fine Chemicals (KF1603, KF1705).

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