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Bacterial Aggregation Assay in the Presence of Cyclic Lipopeptides
环脂肽存在情况下细菌聚集实验   

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Applied and Environmental Microbiology
Jun 2017

Abstract

Lipopeptides is an important class of biosurfactants having antimicrobial and anti-adhesive activity against pathogenic bacteria. These include surfactin, fengycin, iturin, bacillomycin, mycosubtilin, lichenysin, and pumilacidin (Arima et al., 1968; Naruse et al., 1990; Yakimov et al., 1995; Steller and Vater, 2000; Roongsawang et al., 2002; Vater et al., 2002). To date, none of these lipopeptides have been reported to possess any anti-motility activity. We isolated, purified and characterized two novel cyclic lipopeptides (CLPs) from Bacillus sp. 176 using high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. CLPs dramatically suppress the motility of pathogenic bacterium Vibrio alginolyticus 178, and promote cellular aggregation without inducing cell death. Cell aggregation assay was performed with the modification according to methods described by Dalili for anti-biofilm assay (Dalili et al., 2015). In future, this assay can be adapted to test both the cell aggregation and anti-biofilm activity of lipopeptide-like active substances derived from bacteria.

Keywords: Cyclic lipopeptides (环脂肽), Cell aggregation (细胞聚集), Vibrio alginolyticus (溶藻弧菌), Bacillus sp. 176 (芽孢杆菌176), Anti-motility (抗肠蠕动), Anti-biofilm (抗生物膜)

Background

Overuse of broad-spectrum antibiotics and the accompanying proliferation of drug-resistant bacteria have stimulated efforts to develop environment-friendly biocontrol measures to reduce health hazards and environmental pollution (Nam et al., 2016; Sajitha et al., 2016). In recent years, the anti-microbial properties of biological surfactants have been increasingly recognized and harnessed for antibacterial, antifungal, and antiviral applications (Cameotra and Makkar, 2004; Singh and Cameotra, 2004; Rodrigues et al., 2006). Lipopeptides are the most widely reported class of biosurfactants having antimicrobial and 100 anti-adhesive activity against pathogenic bacteria, due to the amphipathic nature of their peptide and fatty acid components (Das et al., 2008; Dalili et al., 2015). In this study, two cyclic lipopeptides (CLPs) derived from a competing bacterium (Bacillus sp. 176) are found to inhibit the motility and promote the aggregation of V. alginolyticus 178. We purified and characterized the active anti-motility compounds and determined their structural and functional properties. In order to explore the mechanism of action of the CLPs, their impact on cell aggregation, adherence, and the expression of flagellar assembly components in V. alginolyticus were also investigated.

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog numbers: T-200-Y-STK , T-300-L-R , T-1000-B )
  2. 15 ml culture tube (Bomei, catalog number: SGJS15ML )
  3. Flat bottom 96-well microtiter plate (Corning, catalog number: 3628 )
  4. Coverslips (CITOTEST LABWARE MANUFACTURING, catalog number: 80340-1130 )
  5. Bacterial strains (Identified and stored in our lab)
    1. V. alginolyticus 178
    2. V. anguillarum
    3. V. splendidus
    4. V. vulnificus
    5. Pseudomonas aeruginosa
    6. P. stutzeri
    7. Staphylococcus aureus
    8. Bacillus sp. 176
    9. B. subtilis
  6. Methanol (Sinopharm Chemical Reagent, catalog number: 10014108 )
  7. Ethanol (Sinopharm Chemical Reagent, catalog number: 10009259 )
  8. Peptone (Solarbio, catalog number: P8450 )
  9. Tryptone (OXOID, catalog number: LP0042 )
  10. Yeast extract (OXOID, catalog number: LP0021 )
  11. Agar powder (Solarbio, catalog number: A8190 )
  12. Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 10019318 )
  13. Crystal violet (Sinopharm Chemical Reagent, catalog number: 71012314 )
  14. Glacial acetic acid (Sinopharm Chemical Reagent, catalog number: 10000218 )
  15. Gelatin (Solarbio, catalog number: G8060 )
  16. 25% glutaraldehyde solution (Sinopharm Chemical Reagent, catalog number: 30092436 )
  17. Dimethyl sulfoxide (DMSO) (MP Biomedicals, catalog number: 02196055 )
  18. Saline LB broth (see Recipes)
  19. LB broth (see Recipes)
  20. 1% (w/v) solution of crystal violet (see Recipes)
  21. 30% (v/v) acetic acid (see Recipes)
  22. Modified 2216E broth (see Recipes)
  23. 1% (w/v) gelatin solution (see Recipes)
  24. 5% glutaraldehyde solution (see Recipes)
  25. Sterile saline solution (see Recipes)
  26. 10 mg/ml CLPs (see Recipes)

Equipment

  1. 1-10 μl pipettor (Gilson, model: P10N )
  2. 20-200 μl pipettor (Gilson, model: P200N )
  3. 100-1,000 μl pipettor (Gilson, model: P1000N )
  4. Autoclave sterilizer (Zealway Instrument, model: GI80TW )
  5. Constant temperature shaker (CRYSTAL, model: IS-RDS3 )
  6. Centrifuge (Eppendorf, model: 5418 R )
  7. SYNERGY-H1 microplate reader (BioTek Instruments, model: Synergy H1 )
  8. Scanning electron microscope (SEM) (Hitachi, model: S-3400N )
  9. Transmission electron microscope (TEM) (Hitachi, model: H-7650 )
  10. Biological safety cabinet (Heal Force, model: HFsafe 900LC )

Procedure

  1. Bacterial strains culture
    1. Single colonies of bacterial strains used in this protocol, including V. alginolyticus 178, V. anguillarum, V. splendidus, V. vulnificus, Pseudomonas aeruginosa, P. stutzeri, Staphylococcus aureus, Bacillus sp. 176 and B. subtilis, were selected from their pure culture plates.
    2. V. alginolyticus 178, V. anguillarum, V. splendidus, V. vulnificus, Pseudomonas aeruginosa, P. stutzeri and Bacillus sp. 176 were cultured in 5 ml saline Luria-Bertani (LB) broth (see Recipes) in tubes at 28 °C overnight with shaking at 170 rpm.
    3. Staphylococcus aureus and B. subtilis were cultured in 5 ml LB broth in tubes at 37 °C overnight with shaking at 170 rpm.

  2. Aggregation assay of V. alginolyticus 178 in culture tubes
    1. After overnight culture, dilute the cell suspension of V. alginolyticus 178 at 1:100 with saline LB broth.
    2. Prepare six sterilized 15 ml culture tubes.
    3. Add 3 ml bacterial suspension into each tube.
    4. Treat the cells with 90 μl (10 mg/ml) CLPs with a final concentration of 300 μg/ml or same volume DMSO, respectively, and incubate statically at 28 °C for 24 h after homogenization (Figure 1A).


      Figure 1. Experimental design of V. alginolyticus 178 aggregation in culture tubes and 96-well plates. A. Aggregation of V. alginolyticus 178 was observed in culture tubes after CLPs treatment. B. Schematic representation of the steps in aggregation assay using 96-well plates (No. 1 well contains untreated V. alginolyticus 178; No. 2 well contains CLPs treated V. alginolyticus 178).

  3. Aggregation assay of V. alginolyticus 178 in 96-well microtiter plate
    1. Add 200 μl diluted bacterial suspension into each well of the flat bottom 96-well microtiter plate.
    2. In experiment group, add 6 μl CLPs (10 mg/ml) with a final concentration of 300 μg/ml in each well, and homogenize with bacterial suspension.
    3. Wells containing V. alginolyticus 178 cell suspension without treatment, or with DMSO treatment, are employed as controls.
    4. Five replicate wells for each treatment, and incubate the plate at 28 °C for 24 h.
    5. After incubation, discard the planktonic cells carefully with a pipettor and wash the aggregated cells in each well three times with sterile saline.
    6. Fix aggregated cells with 200 μl of methanol (99% purity) per well statically for 15 min, and empty the plates using a pipettor and leave to dry.
    7. Then stain the contents of the wells with 200 μl of a 1% (w/v) crystal violet (see Recipes) solution for 10 min at room temperature.
    8. Rinse out crystal violet with a pipettor; air-dry the plates after wash with sterile deionized water, and resolubilize the dye bound to the aggregated cells in 200 μl of 30% (v/v) acetic acid (see Recipes) solution.
    9. Measure the absorbance of each well in a SYNERGY-H1 microplate reader (BioTek, USA) at 595 nm using 30% acetic acid as the blank (Figure 1B).

  4. Quantitative assay of CLPs’ activity
    1. Add 200 μl diluted bacterial suspension into each well of the flat bottom 96-well microtiter plate.
    2. Add 6 μl CLPs at different concentration (25, 50, 100, 200, 300 μg/ml) into different wells, respectively.
    3. Wells containing V. alginolyticus 178 cell suspension without treatment, or with DMSO treatment, are employed as controls.
    4. Operate the other procedures as described above in Steps C4-C9 (Figure 2).


      Figure 2. Quantitative cell aggregation assay at different concentrations of CLPs

  5. Scanning electron microscope observation
    1. Dilute overnight cultured cell of V. alginolyticus 178 at 1:100 into 5 ml fresh modified 2216E medium (see Recipes) and culture for another 3 h to OD600 0.2-0.3 with shaking at 170 rpm.
    2. Add the cell suspension with 50 μl 50 or 100 μg/ml CLPs or DMSO for additional 3 h.
    3. Centrifuge the cells at 1,400 x g for 4 min at room temperature, and resuspend the cells with a sterilized saline solution.
    4. Drop the resuspended bacterial cells on sterilized coverslips (enveloped by 1% gelation solution, see Recipes), respectively.
    5. Dry coverslips at room temperature.
    6. Fix samples with a 5% glutaraldehyde solution (see Recipes) for 1 h.
    7. Wash with sterile saline solution (see Recipes).
    8. Dehydrate samples in a successively graded ethanol series (50%, 60%, 70%, 80%, 90% and 100% ethanol). Incubate the samples for 10 min in each grade ethanol solution. After the final incubation in 100% ethanol, the samples are ready for observation under scanning electron microscope.
      Note: You can stop when the samples are placed in 80% ethanol.
    9. Observe the samples under a scanning electron microscope (Figure 3).


      Figure 3. SEM images of V. alginolyticus 178 cells following treatment with CLPs. V. alginolyticus 178 cell morphology without any treatment (A), with DMSO treatment (B), with 50 μg/ml CLPs (C), and with 100 μg/ml CLP treatment (D).

  6. Transmission electron microscope observation
    1. Perform the procedures as described above in Steps E1-E3.
    2. Fix samples with a 5% glutaraldehyde solution for 1 h.
    3. Drop samples on copper grids, and dried at room temperature.
    4. Observe the samples under a transmission electron microscope (Figure 4).


      Figure 4. TEM images of V. alginolyticus 178 cells following treatment with CLPs. V. alginolyticus 178 cell morphology without any treatment (A), with DMSO treatment (B), with 50 μg/ml CLPs (C), and with 100 μg/ml CLP treatment (D). The scale bars are 2 μm.

  7. The spectrum of action of the CLPs
    1. After overnight culture, dilute the cell suspension of V. anguillarum, V. splendidus, V. vulnificus, Pseudomonas aeruginosa, P. stutzeri, Staphylococcus aureus, Bacillus sp. 176 and B. subtilis at 1:100 with saline LB broth or LB broth, respectively.
    2. Operate the other procedures as described above in Steps C2-C9 (Figure 5).


      Figure 5. Action spectrum assays of CLPs. Blank: untreated strains; CLPs: CLPs treated strains.

Data analysis

All data were analyzed by the Statistical Package for Social Sciences (SPSS) 18.0 software. The statistically significant differences among groups were calculated using one-way analysis of variance (one-way ANOVA) followed by a post hoc multiple-comparisons (Tukey’s) test.

Notes

  1. To avoid other bacteria contamination, microbial operations must be carried out under aseptic conditions.
  2. All bacterial mediums and reagent solutions are sure to be prepared freshly and sterilized by autoclaving.
  3. It is necessary to homogenize the cell suspension with CLPs before incubation.
  4. Notice that the operation in discarding the planktonic cells and washing the aggregated cells with pipettor must be careful and gentle.

Recipes

  1. Saline LB broth (1 L)
    10 g peptone
    5 g yeast extract
    1 L filtered seawater
    pH 7.4-7.6
    Sterilize by autoclaving at 121 °C for 20 min, and store at 4 °C
  2. LB broth (1 L)
    10 g peptone
    10 g NaCl
    5 g yeast extract
    1 L deionized water
    pH 7.4-7.6
    Sterilize by autoclaving at 121 °C for 20 min, and store at 4 °C
  3. 1% (w/v) solution of crystal violet (100 ml)
    1 g crystal violet
    100 ml sterile water
  4. 30% (v/v) acetic acid (100 ml)
    30 ml glacial acetic acid
    70 ml sterile water
  5. Modified 2216E broth (1 L)
    5 g tryptone
    1 g yeast extract
    1 L filtered seawater
    pH 7.4-7.6
    Sterilize by autoclaving at 121 °C for 20 min, and store at 4 °C
  6. 1% (w/v) gelatin solution (10 ml)
    0.1 g gelatin
    10 ml deionized water
    Sterilize by autoclaving at 121 °C for 20 min, and store at 4 °C
  7. 5% glutaraldehyde solution (10 ml)
    2 ml 25% glutaraldehyde solution
    8 ml sterile water
  8. Sterile saline solution (100 ml)
    0.85 g NaCl
    100 ml deionized water
    Sterilize by autoclaving at 121 °C for 20 min, and store at 4 °C
  9. 10 mg/ml CLPs (1 ml)
    10 mg CLPs
    1 ml DMSO

Acknowledgments

This work was supported by Natural science outstanding youth fund of Shandong Province (JQ201607), Tai Shan Scholar Foundation of Shandong Province, AoShan Talents Program supported by Qingdao National Laboratory for Marine Science and Technology (No. 2015ASTP), ‘100-Talent Project’ of the Chinese Academy of Sciences to Chaomin Sun, and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA11030201) to Dechao Zhang. We also really appreciate Professor Nasrin Samadi and Dina Dalili, from Tehran University of Medical Sciences, for their method in study ‘Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity’. And the authors declare that they have no competing interests.

References

  1. Arima, K., Kakinuma, A. and Tamura, G. (1968). Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochem Biophys Res Commun 31(3): 488-494.
  2. Cameotra, S. S. and Makkar, R. S. (2004). Recent applications of biosurfactants as biological and immunological molecules. Curr Opin Microbiol 7(3): 262-266.
  3. Dalili, D., Amini, M., Faramarzi, M. A., Fazeli, M. R., Khoshayand, M. R. and Samadi, N. (2015). Isolation and structural characterization of Coryxin, a novel cyclic lipopeptide from Corynebacterium xerosis NS5 having emulsifying and anti-biofilm activity. Colloids Surf B Biointerfaces 135: 425-432.
  4. Das, P., Mukherjee, S. and Sen, R. (2008). Antimicrobial potential of a lipopeptide biosurfactant derived from a marine Bacillus circulans. J Appl Microbiol 104(6): 1675-1684.
  5. Nam, H. S., Yang, H. J., Oh, B. J., Anderson, A. J. and Kim, Y. C. (2016). Biological control potential of Bacillus amyloliquefaciens KB3 isolated from the feces of Allomyrina dichotoma larvae. Plant Pathol J 32(3): 273-280.
  6. Naruse, N., Tenmyo, O., Kobaru, S., Kamei, H., Miyaki, T., Konishi, M. and Oki, T. (1990). Pumilacidin, a complex of new antiviral antibiotics. Production, isolation, chemical properties, structure and biological activity. J Antibiot (Tokyo) 43(3): 267-280.
  7. Rodrigues, L., Banat, I. M., Teixeira, J. and Oliveira, R. (2006). Biosurfactants: potential applications in medicine. J Antimicrob Chemother 57(4): 609-618.
  8. Roongsawang, N., Thaniyavarn, J., Thaniyavarn, S., Kameyama, T., Haruki, M., Imanaka, T., Morikawa, M. and Kanaya, S. (2002). Isolation and characterization of a halotolerant Bacillus subtilis BBK-1 which produces three kinds of lipopeptides: bacillomycin L, plipastatin, and surfactin. Extremophiles 6(6): 499-506.
  9. Sajitha, K. L., Dev, S. A. and Maria Florence, E. J. (2016). Identification and characterization of lipopeptides from Bacillus subtilis B1 against sapstain fungus of rubberwood through MALDI-TOF-MS and RT-PCR. Curr Microbiol 73(1): 46-53.
  10. Singh, P. and Cameotra, S. S. (2004). Potential applications of microbial surfactants in biomedical sciences. Trends Biotechnol 22(3): 142-146.
  11. Steller, S. and Vater, J. (2000). Purification of the fengycin synthetase multienzyme system from Bacillus subtilis b213. J Chromatogr B Biomed Sci Appl 737(1-2): 267-275.
  12. Vater, J., Kablitz, B., Wilde, C., Franke, P., Mehta, N. and Cameotra, S. S. (2002). Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol 68(12): 6210-6219.
  13. Yakimov, M. M., Timmis, K. N., Wray, V. and Fredrickson, H. L. (1995). Characterization of a new lipopeptide surfactant produced by thermotolerant and halotolerant subsurface Bacillus licheniformis BAS50. Appl Environ Microbiol 61(5): 1706-1713.

简介

脂肽是一类重要的生物表面活性剂,对致病菌具有抗菌和抗粘连活性。这些包括表面活性肽,fengycin,伊库菌素,杆菌霉素,mycosubtilin,地衣素和pumilacidin(Arima等人,1968; Naruse等人,1990; Yakimov等人1995; Steller和Vater,2000; Roongsawang等人,2002; Vater等人,2002)。迄今为止,这些脂肽都没有被报道具有任何抗运动活性。我们从芽孢杆菌分离,纯化和鉴定了两种新的环状脂肽(CLPs)。 176使用高效液相色谱,质谱和核磁共振光谱。 CLPs极大地抑制致病性溶藻弧菌的运动,并促进细胞聚集而不诱导细胞死亡。根据Dalili所述用于抗生物膜测定的方法进行细胞聚集测定(Dalili等人,2015)。将来,该测定法可以适用于测试来自细菌的脂肽样活性物质的细胞聚集和抗生物膜活性。

【背景】过度使用广谱抗生素和随之而来的抗药性细菌的增殖刺激了开发环境友好型生物防治措施的努力,以减少健康危害和环境污染(Nam等人,2016; Sajitha >等。,2016)。近年来,生物表面活性剂的抗微生物特性已被越来越多地用于抗菌,抗真菌和抗病毒的应用(Cameotra和Makkar,2004; Singh和Cameotra,2004; Rodrigues等人 ,2006)。由于其肽和脂肪酸组分的两亲性质,脂肽是最广泛报道的具有抗微生物和抗粘附活性的生物表面活性剂类(Das等人,2008; Dalili等人,等人,2015年)。在这项研究中,发现来源于竞争性细菌(芽孢杆菌176)的两种环状脂肽(CLPs)抑制运动性并促进Vv的聚集。溶藻酶178.我们纯化和表征了活性抗蠕动化合物并确定了它们的结构和功能特性。为了探讨CLPs的作用机制,它们对细胞聚集,粘附和鞭毛组装成分表达的影响。也研究了溶藻弧菌。

关键字:环脂肽, 细胞聚集, 溶藻弧菌, 芽孢杆菌176, 抗肠蠕动, 抗生物膜

材料和试剂

  1. 移液器吸头(Corning,Axygen ,目录号:T-200-Y-STK,T-300-L-R,T-1000-B)
  2. 15毫升培养管(博美,目录号:SGJS15ML)
  3. 平底96孔微量滴定板(Corning,目录号:3628)
  4. 盖帽(CITOTEST LABWARE MANUFACTURING,产品目录编号:80340-1130)
  5. 细菌菌株(鉴定和存储在我们的实验室)
    1. 诉alginolyticus 178
    2. 诉anguillarum
    3. 诉灿烂的
    4. 诉创伤
    5. 铜绿假单胞菌
    6. P上。 stutzeri
    7. 金黄色葡萄球菌
    8. 芽孢杆菌属176
    9. B中。 subtilis
  6. 甲醇(国药集团化学试剂,目录号:10014108)
  7. 乙醇(国药集团化学试剂,产品目录号:10009259)
  8. 蛋白胨(Solarbio,目录号:P8450)
  9. 胰蛋白胨(OXOID,目录号:LP0042)
  10. 酵母提取物(OXOID,目录号:LP0021)
  11. 琼脂粉(Solarbio,目录号:A8190)
  12. 氯化钠(NaCl)(国药集团化学试剂,目录号:10019318)
  13. 结晶紫(国药集团化学试剂,目录号:71012314)
  14. 冰醋酸(国药集团化学试剂,目录号:10000218)
  15. 明胶(Solarbio,目录号:G8060)
  16. 25%戊二醛溶液(国药集团化学试剂,产品目录号:30092436)
  17. 二甲基亚砜(DMSO)(MP Biomedicals,目录号:02196055)
  18. 盐水LB肉汤(见食谱)
  19. LB肉汤(见食谱)
  20. 1%(w / v)结晶紫溶液(见食谱)
  21. 30%(v / v)乙酸(见配方)
  22. 改良2216E肉汤(见食谱)
  23. 1%(w / v)明胶溶液(见食谱)
  24. 5%戊二醛溶液(见食谱)
  25. 无菌生理盐水(见食谱)
  26. 10毫克/毫升CLPs(见食谱)

设备

  1. 1-10微升移液器(Gilson,型号:P10N)
  2. 20-200μl移液器(Gilson,型号:P200N)
  3. 100-1,000μl移液器(Gilson,型号:P1000N)
  4. 高压灭菌器(Zealway仪器,型号:GI80TW)
  5. 恒温摇床(CRYSTAL,型号:IS-RDS3)
  6. 离心机(Eppendorf,型号:5418 R)
  7. SYNERGY-H1酶标仪(BioTek Instruments,型号:Synergy H1)
  8. 扫描电子显微镜(SEM)(日立,型号:S-3400N)
  9. 透射电子显微镜(TEM)(日立,型号:H-7650)
  10. 生物安全柜(Heal Force,型号:HFsafe 900LC)

程序

  1. 细菌菌株培养
    1. 在这个协议中使用的细菌菌株的单菌落,包括溶藻弧菌(V. alginolyticus)178, anguillarum , V。灿烂的, V。创伤弧菌,铜绿假单胞菌, p。 stutzeri , Staphylococcus aureus , Bacillus sp。 176和。从他们纯粹的培养皿中选择了枯草芽孢杆菌(subtilis)。
    2. 诉溶藻弧菌178,鳗弧菌V.vir。 ,金黄色葡萄球菌(V. vulnificus),铜绿假单胞菌(Pseudomonas aeruginosa),金黄色葡萄球菌stutzeri 和 Bacillus sp。 176在含有Luria-Bertani(LB)的5毫升生理盐水(LB)肉汤培养基(见配方)中于28℃培养过夜,摇动速度为170转/分。
    3. 金黄色葡萄球菌和 B。枯草芽孢杆菌在5ml LB培养基中于37℃在管中振荡培养过夜,在170rpm下振荡。

  2. 溶藻弧菌178在培养管中的聚集测定
    1. 过夜培养后,稀释 V的细胞悬液。
      用生理盐水LB肉汤以1:100溶解溶藻弧菌178
    2. 准备六个无菌15毫升培养管。

    3. 每个管中加入3毫升细菌悬液
    4. 用终浓度为300μg/ ml或相同体积DMSO的90μl(10 mg / ml)CLPs分别处理细胞,匀浆后在28℃静置培养24 h(图1A)。


      图1. V的实验设计A.溶藻弧菌在培养管和96孔板中的聚集A.在CLPs处理后在培养管中观察到溶藻弧菌178。 B.使用96孔板进行聚集测定的步骤的示意图(1号孔含有未经处理的溶藻弧菌178号; 2号孔含有经溶藻弧菌处理的溶藻弧菌) > 178)。

  3. V的聚集测定。在96孔微量滴定板中溶藻弧菌178

    1. 加入200μl稀释的细菌悬液到平底96孔微量滴定板的每个孔中
    2. 在实验组中,每孔加入终浓度为300μg/ ml的6μlCLPs(10 mg / ml),并与细菌悬液均质化。
    3. 包含 V的井。采用未经处理的溶藻弧菌178细胞悬液或用DMSO处理,作为对照。
    4. 每个处理5个重复孔,并在28°C孵育24小时。
    5. 孵育后,用移液器小心丢弃浮游细胞,并用无菌生理盐水洗涤每个孔中的聚集细胞三次。
    6. 用200μl甲醇(99%纯度)固定聚集的细胞,静置15分钟,用移液器清空培养板,放置干燥。
    7. 然后在室温下用200μl1%(w / v)结晶紫(见配方)溶液染色孔内容物10分钟。
    8. 用移液器冲洗结晶紫;用无菌去离子水冲洗后,用200μl30%(v / v)醋酸溶液(参见食谱)溶解后将板上的染料再溶解于聚集的细胞中。
    9. 用SYNERGY-H1酶标仪(BioTek,USA)在595 nm处使用30%乙酸作为空白(图1B),测量每个孔的吸光度。

  4. CLPs活性的定量测定

    1. 加入200μl稀释的细菌悬液到平底96孔微量滴定板的每个孔中
    2. 分别加入不同浓度(25,50,100,200,300μg/ ml)的6μlCLPs到不同的孔中。
    3. 包含 V的井。使用溶藻弧菌178不经处理的细胞悬浮液或用DMSO处理作为对照。
    4. 按照上述步骤C4-C9(图2)所述操作其他程序。


      图2.不同浓度CLPs的定量细胞聚集分析

  5. 扫描电镜观察
    1. 稀释过夜的培养细胞的V。溶于1ml / ml的溶藻弧菌178溶于5ml新鲜的改良的2216E培养基中(参见食谱),并以170rpm振荡培养另外3小时至OD 600-6.2-0.3。

    2. 添加50μl50或100μg/ ml CLPs或DMSO的细胞悬液3小时。
    3. 在室温下将细胞在1,400×g下离心4分钟,并用无菌盐水溶液重悬细胞。
    4. 将重新悬浮的细菌细胞分别放在灭菌的盖玻片上(用1%凝胶溶液包封,参见食谱)。
    5. 在室温下干燥盖玻片。
    6. 用5%戊二醛溶液固定样品(见食谱)1小时。
    7. 用无菌生理盐水冲洗(见食谱)。
    8. 在连续分级乙醇系列(50%,60%,70%,80%,90%和100%乙醇)中脱水样品。在每个等级的乙醇溶液中孵育样品10分钟。最后在100%乙醇中孵育后,样品准备在扫描电子显微镜下观察。
      注意:当样品放入80%乙醇中时,您可以停止。
    9. 在扫描电子显微镜下观察样品(图3)。


      图3. V的SEM图像。用CLPs处理后的溶藻弧菌178细胞。 (A),用DMSO处理(B),用50μg/ ml CLPs(C)和用100μg/ ml CLP处理(D)处理溶藻弧菌178细胞形态。
  6. 透射电镜观察
    1. 执行上述步骤E1-E3中的步骤。
    2. 用5%戊二醛溶液固定样品1小时。
    3. 将样品放在铜网上,并在室温下干燥。
    4. 透射电镜下观察样品(图4)。


      图4. V的TEM图像。用CLPs处理后的溶藻弧菌178细胞。 诉(A),用DMSO处理(B),用50μg/ ml CLPs(C)和用100μg/ ml CLP处理(D)处理溶藻弧菌178细胞形态。比例尺是2微米。

  7. CLP的行动范围
    1. 过夜培养后,稀释 V的细胞悬液。 anguillarum , V。灿烂的, V。创伤弧菌,铜绿假单胞菌, p。 Stutzeri , Staphylococcus aureus , 176和。用1%盐水LB肉汤或LB肉汤分别加入枯草芽孢杆菌(Bacillus subtilis)。
    2. 按照上述步骤C2-C9(图5)操作其他程序。


      图5. CLPs的作用谱分析。空白:未处理的菌株; CLPs:CLPs治疗的菌株。

数据分析

所有数据均由社会科学统计软件包(SPSS)18.0软件进行分析。使用单因素方差分析(one-way ANOVA),然后进行事后多重比较(Tukey's)检验,计算组间统计学显着性差异。

笔记

  1. 为避免其他细菌污染,微生物操作必须在无菌条件下进行。
  2. 所有的细菌培养基和试剂溶液都必须新鲜制备,并通过高压灭菌来灭菌。

  3. 在孵化之前,需要用CLPs匀浆细胞悬液
  4. 请注意,丢弃浮游细胞和用移液器清洗聚集细胞的操作必须小心谨慎。

食谱

  1. 盐水LB肉汤(1升)
    10克蛋白胨
    5克酵母提取物
    1升过滤海水
    pH 7.4-7.6

    在121°C高压灭菌20分钟,然后储存在4°C
  2. LB肉汤(1升)
    10克蛋白胨
    10克NaCl
    5克酵母提取物
    1升去离子水
    pH 7.4-7.6

    在121°C高压灭菌20分钟,然后储存在4°C
  3. 1%(w / v)结晶紫溶液(100 ml)
    1克结晶紫
    100毫升无菌水
  4. 30%(v / v)乙酸(100ml)
    30毫升冰醋酸
    70毫升无菌水
  5. 改性2216E肉汤(1升)
    5克胰蛋白胨
    1克酵母提取物
    1升过滤海水
    pH 7.4-7.6

    在121°C高压灭菌20分钟,然后储存在4°C
  6. 1%(w / v)明胶溶液(10毫升)
    0.1克明胶
    10毫升去离子水

    在121°C高压灭菌20分钟,然后储存在4°C
  7. 5%戊二醛溶液(10毫升)
    2毫升25%戊二醛溶液
    8毫升无菌水
  8. 无菌生理盐水(100毫升)
    0.85克NaCl 100毫升去离子水

    在121°C高压灭菌20分钟,然后储存在4°C
  9. 10毫克/毫升CLPs(1毫升)
    10毫克CLPs
    1毫升DMSO

致谢

这项工作得到了山东省自然科学优秀青年基金(JQ201607),山东省泰山学者基金,青岛海洋科学与技术国家实验室(2015ASAS)支持的“奥山人才计划”,“百人计划”中国科学院向孙敬民和中国科学院战略重点研究项目(XDA11030201)致张德超。我们也非常感谢来自德黑兰医科大学的Nasrin Samadi教授和Dina Dalili教授的研究方法“Isolation and structural characterization of Coryxin,a novel cyclic lipopeptide from from Corynebacterium xerosis NS5 having emulsification and anti生物膜活性“。作者声明他们没有竞争利益。

参考

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引用:Xiu, P., Liu, R., Zhang, D. and Sun, C. (2018). Bacterial Aggregation Assay in the Presence of Cyclic Lipopeptides. Bio-protocol 8(1): e2686. DOI: 10.21769/BioProtoc.2686.
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