CN106706693A - Metabonomics analysis method for quorum sensing inhibition mechanism - Google Patents
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Abstract
The invention discloses a metabonomics analysis method for a quorum sensing inhibition mechanism. Through extraction of bacterium metabolites and <1>H-NMR test, R language is adopted for carrying out orthogonal signal correction-partial least square method discrimination analysis on the <1>H-NMR spectrogram, screening out differentially expressed metabolites, calculating metabolite changing rates of a quorum sensing inhibition group and a control group, selecting the changed metabolites related to quorum sensing and carrying out metabolism pathway analysis, identifying the changed metabolism pathway and analyzing a bacterium quorum sensing inhibition mechanism of a quorum sensing inhibitor. The metabonomics analysis method disclosed by the invention has the advantages that metabonomics is adopted, the total metabolic state of an organism is dynamically and integrally described, and the action mechanism of the quorum sensing inhibitor can be effectively analyzed.
Description
Technical field
The invention belongs to metabolism group application field, and in particular to a kind of metabonomic analysis of quorum sensing suppression mechanism
Method.
Background technology
Intervention school-based realizes it to pathogenic regulation and control by mediating the expression of Disease-causing gene, in addition quorum sensing
System also regulates and controls the formation of pathogen biofilm, and the formation of biofilm is also a major reason of bacterial drug resistance.
Having returned now various quorum sensing inhibitor can control the formation of Pathogenic and biofilm.
Metabolism group is after genomics, transcription group and newly-developed gets up after proteomics a subject,
It is a science that organism metabolism is studied in integral level.Metabolism group is to a certain biological or cell in a specific life
In reason period all low-molecular-weights metabolite simultaneously carry out qualitative and quantitative analysis, by analyze cell, tissue, organ or
Many micromolecular compounds in person's biofluid capture biochemical reaction, and the metabolism of application message technical definition is special
Levy.Metabolin reflects the environment residing for cell, the nutritional status with cell, the effect of medicine and environmental contaminants, Yi Jiqi
The influence of its extraneous factor is closely related.
Although many researchs make great efforts to probe into the mechanism of action of quorum sensing inhibitor now, on them to generation
The still rare report of the influence thanked.In numerous analysis methods of metabolism group application, such as LC-MS, gas chromatography mass spectrometry and nuclear-magnetism
Resonance (NMR), nuclear magnetic resonance it is most widely used because its preparation of samples is simple, analysis process without destructiveness, soon
Speed, while abundant structural information can be obtained.It is based on1The metabolism group of H-NMR, with reference to multi-variate statistical analysis, by with
In overall metabolic condition (Yangfang Ye, the et al.Global Metabolomic Responses of of research bacterium
Escherichia coli to Heat Stress.J.Proteome Res.2012,11,2559-2566).But utilize generation
Xie Zuxue come study compound suppress quorum sensing mechanism do not have been reported that also.
The content of the invention
It is an object of the invention to provide a kind of metabonomic analysis methods of quorum sensing suppression mechanism.
The technical scheme is that：
A kind of metabonomic analysis methods of quorum sensing suppression mechanism, using being based on1The metabolism group method pair of H-NMR
Quorum sensing suppression mechanism is analyzed, and comprises the following steps：
Step 1, the culture of bacterial strain：The seed liquor of the bacterial strain with intervention school-based is inoculated into culture medium, is added
Quorum sensing inhibitor solution, with isometric solvent as a control group, Amplification Culture；
Step 2, extracts metabolin：After culture terminates, bacterium solution is placed in of short duration hatching on ice, bacterial strain is stopped metabolism, it is low
Warm high speed centrifugation obtains thalline, extracts metabolin；
Step 3,1H-NMR is tested：Dry extract is dissolved in deuterated reagent, is carried out1H-NMR is tested, and is obtained1H-
NMR spectra；
Step 4, data processing：Will1H-NMR spectrum phase calibration, adjustment baseline, and it is zero point position to adjust interior target peak
Move, specific metabolin is determined according to characteristic peak；
Step 5, using R language pair1H-NMR spectrum carries out orthogonal signalling correction-PLS discriminant analysis (OSC-
PLS-DA), the metabolin of differential expression between quorum sensing suppression group and control group is filtered out, selection senses system with bacterial community
The associated P values of system are less than 0.05 and multiplying power change is more than 1.1 or the metabolin less than 0.9 carries out metabolic pathway analysis.
Compared with prior art, remarkable advantage of the invention is：
1st, the mechanism of action of quorum sensing inhibitor is probed into the method for metabolism group first；
2nd, with being based on1The metabolism group method analysis metabolin of H-NMR, preparation of samples is simple, analysis process to sample not
With destructiveness, at a high speed, and can obtain not having structural information devious, abundant；
3rd, the metabolic alterations of bacterium can dynamically, be comprehensively observed with the method for metabolism group, obtains bacterium complete
Metabolic information.
Brief description of the drawings
Fig. 1 is the typical case of pseudomonas aeruginosa PAO1 resveratrols treatment group and control group extract in embodiment 31H-
NMR spectra.
Fig. 2 is the shot chart of the OSC-PLS-DA in embodiment 4.
Fig. 3 is the S curve figure of the OSC-PLS-DA in embodiment 4.
Fig. 4 is the load diagram of the OSC-PLS-DA in embodiment 4.
Specific embodiment
With reference to embodiment and accompanying drawing, the invention will be further described.
Resveratrol can suppress the intervention school-based of pseudomonas aeruginosa PAO1 as quorum sensing inhibitor, at this
It is quorum sensing inhibitor with resveratrol in inventive embodiments, is with intervention school-based with pseudomonas aeruginosa PAO1
Bacterial strain, the present invention is elaborated.
Embodiment 1：The extraction of pseudomonas aeruginosa PAO1 metabolins and the preparation of nuclear-magnetism sample
The single bacterium of picking pseudomonas aeruginosa PAO1 is dropped down onto in NB culture mediums, 37 DEG C, 150rpm overnight incubations.To overnight train
Foster bacterium solution 1: 1000 is diluted in the conical flask equipped with NB culture mediums, adds resveratrol to make its final concentration of 400 μM, waits body
Long-pending DMSO is put into shaking table as control after being well mixed, 37 DEG C, 180rpm 16~18h of culture, 13 weights of every group of setting
It is multiple.After cultured bacterium solution is placed on into of short duration hatching on ice, 4 DEG C, 10000rpm centrifugation 8min, abandoning supernatant obtain thalline.
After thalline is washed into three times with precooled PBS, it is transferred in a new 10mL centrifuge tube.Addition 3.8mL precooled methyl alcohol/
Water (1/0.9；V/v) extract solution, on ice, interval ultrasound 5min is extracted.4mL chloroforms are added, after being fully vortexed, 4
DEG C, 10000rpm centrifugation 8min.Water is mutually transferred in a new 5mL centrifuge tube, vacuum drying removal methyl alcohol is put into freezing
At least 24h is freezed in drying machine.Dry extract is dissolved in 550 μ L and contains 10%D2The phosphate of O and 0.02%TSP delays
In fliud flushing (0.1M, pH 7.4), 10min is centrifuged.Supernatant is drawn to be transferred in the nuclear magnetic tube of 5mm.
Embodiment 2：1H-NMR is tested
The metabolin sample extracted in bacterium solution1H-NMR tests are in Bruker Av500MHz Liquid NMR spectrometers
In carry out.Collecting temperature is 298K, uses D2O locks field, the peak of internal standard TSP as zero point chemical shift reference (1H,δ0.00).Make
" cpmg " (Call-Purcell-Meiboom-Gill) sequence acquisition with suppression aquapulse is one-dimensional1H-NMR。1H-NMR spectrum FID
Accumulative frequency is 128 times, and sampling number is 32K, and spectrum width is set to 10,330Hz, and acquisition time is 3.27s, relaxation time
It is 3.0s.Before Fourier transformation, will be all one-dimensional1The FID of H-NMR spectrum is multiplied by the window index that the broadening factor is 0.5Hz
Function.
Embodiment 3：1The pretreatment of H-NMR data and the ownership of characteristic peak
It is all of1H-NMR spectrum the softwares of Bruker Topspin 3.0 (Brooker Co., Ltd, Karlsruhe,
Germany) on manual phase calibration, adjustment baseline, and adjust the peak of internal standard TSP for null drift (1H,δ0.00).By what is handled well1H-NMR spectrum imports MestReNova softwares (version 8.0.1, Mestrelab Research SL), and exports as ASCII lattice
Formula file.Then the file of ASCII fromat is imported into " R " software (http://cran.r-project.org/) carry out polynary number
Analyze according to statistics.To one-dimensional in 0.2 to 10ppm interval1H-NMR spectrum, according to the unit subsection integral spacing of 0.005ppm
(Buckets) subsection integral, to reduce data points, while removing residual water peak-to-peak signal area (4.5-5.0ppm).Take PQN
(Probability Quotient Normalization) method is to remaining integration1The total wave spectrum area standardizations of H-NMR,
To increase the comparativity of data.Before multi-variables analysis, each group of data is carried out mean center (mean-centered) and
Pareto upscaled (Pareto-scaled) treatment, to balance the signal strength difference that group internal cause sample room concentration causes.
With reference to one-dimensional1H-NMR spectrum and two-dimentional nuclear magnetic spectrogram1H-1H TOCSY and1H-13C HSQC；It is metabolized with reference to comparing
Thing group database is such as：Mankind's metabolism group database (Human Metabolome Database, HMDB, http://
Www.hmdb.ca), MMCD (Madison-Qingdao Metabolomics Consortium Database, http://
mmcd.nmrfam.wisc.edu/)；While utilization software Chenomx NMR suite 7.5 (Chenomx companies, Canada,
Edmonton), and statistics total correlation spectrum (statistical total correlation spectroscopy, STOCSY)
Analysis means pair1H-NMR spectral peaks carry out compound ownership, confirm.Pseudomonas aeruginosa PAO1 resveratrols treatment group and control
The typical case of group extract1H-NMR spectrum is as shown in Figure 2.By referring to metabolomic research database method is compared, identify altogether
Go out 40 kinds of metabolins, including amino acid, organic acid, organic amine and ergastic substances.The details of metabolin, including peak ownership
Be see the table below with multiple change.
The pseudomonas aeruginosa PAO1 of table 1 extracts multiplying power changing value and its P between metabolin attaching information and metabolin group
Value.
Embodiment 4：Statistical analysis
In order to find the group difference between resveratrol treatment group and control group, we are using the orthogonal signalling for having supervision
Correction-PLS discriminant analysis (OSC-PLS-DA) is to experiment1H-NMR data carry out multi-variate statistical analysis.Such as Fig. 2
Shown, OSC-PLS-DA shot charts represent group difference substantially, and with the good goodness of fit.In S- curve maps (Fig. 3),
The point of different colours and shape represents the micro- variable in metabolin：From variable center more away from, illustrate that its contribution is bigger.Colour is carried
In lotus figure (Fig. 4), according to the size of coefficient correlation, from blueness to red, correlation gradually strengthens.By these data analyses we
Obtain, compared with control group, isoleucine, leucine in resveratrol treatment group, valine, acetic acid, acetamide, amber
Acid, aspartic acid, dimethylamine, methyl amimoacetic acid, trimethylamine, monoethanolamine, methyl alcohol, serine and formic acid are dramatically increased, 2- hydroxy-isobutyrics
Acid, N- aceglutamides, glutamic acid, 2-oxoglutaric acid, glycine betaine, fumaric acid and oxipurinol are substantially reduced.
In order to assess whether resveratrol is an effective quorum sensing inhibitor, we suppress colony except paying close attention to it
Outside the ability of induction system, while also to see whether it influences the growth of bacterium.Putrescine reflects the growth conditions of bacterium, and right
Compared according to group, the level of putrescine is not changed in substantially after resveratrol treatment.Illustrating 400 μM of resveratrol does not influence verdigris false
The growth of monad PAO1.
Glycine betaine avoids oxidative damage, maintenance cell membrane knot as the antioxidant in organism in protection organism
The integrality aspect of structure and function plays vital role.Active oxygen will be produced in normal cell metabolism, high
Cell density cause the accumulation of excessive active oxygen.Recent research finds that in pseudomonas aeruginosa oxidative pressure can be gone up
Adjust the expression of the gene of quorum sensing regulation and control.By the antioxidation activity that resveratrol has, it is presumed that resveratrol is logical
Alleviation oxidative pressure is crossed, so as to suppress the quorum sensing of pseudomonas aeruginosa.
Compared with control group, branched-chain amino acid (BCAAs) valine, leucine and different bright ammonia in resveratrol treatment group
Acid content is significantly improved.Branched-chain amino acid, as important substrate and adjuster, is during protein synthesizes in protein synthesis
Essential amino acid.The explanation normal albumen synthesis in pseudomonas aeruginosa PAO1 that increases of branched-chain amino acid is broken down,
This is probably the intervention school-based that pseudomonas aeruginosa PAO1 is have impact on due to the treatment of resveratrol, and intervention school-based
Many virulence factors of regulation and control are all protein.
Resveratrol also results in the disorder of pseudomonas aeruginosa PAO1 energetic supersessions, and this can be from the liter of succinic acid content
Find out in the decline of high and fumaric acid content.Butanedioic acid and fumaric acid are all the intermediates of tricarboxylic acid cycle (TCA), just
Under normal physiological condition, butanedioic acid can be converted into fumaric acid under the catalysis of succinate dehydrogenase.The liter of succinic acid content
The activity that the decline of high and fumaric acid content reflects succinate dehydrogenase is affected, so that tricarboxylic acid cycle is obstructed.
As the most important metabolic pathway of organism production capacity, tricarboxylic acid cycle it is any disorderly all can interfering energy metabolism, cause bacterium
Energy is not enough and dysfunction, finally influences the pathogenic of them.Because the interference of TCA circulations is so as to cause the suppression of aerobic respiration
System, such as anaerobic respiration has to accelerate in interior other modes as compensation, and this can infer from the increase of acetate levels
Obtain, because anaerobic respiration can cause the accumulation of acetic acid.Simultaneously it is reported that the anaerobiosis meeting of pseudomonas aeruginosa
Cause the reduction of cytotoxicity, because the secretion of the virulence factor elastase of quorum sensing regulation and control is suppressed.Therefore, always
For, quorum sensing inhibitor resveratrol suppresses aerobic respiration, such anaerobic respiration by disturbing tricarboxylic acid cycle
Accelerate, so as to suppress the quorum sensing of pseudomonas aeruginosa, such dysfunction causes subtracting for P. aeruginosa bacteria pathogenic
It is weak.
Claims (1)
1. a kind of metabonomic analysis methods of quorum sensing suppression mechanism, it is characterised in that comprise the following steps：
Step 1, the culture of bacterial strain：The seed liquor of the bacterial strain with intervention school-based is inoculated into culture medium, colony is added
Sensing inhibitor solution, with isometric solvent as a control group, Amplification Culture；
Step 2, extracts metabolin：After culture terminates, bacterium solution is placed in of short duration hatching on ice, bacterial strain is stopped metabolism, low temperature is high
Speed centrifugation obtains thalline, extracts metabolin；
Step 3,1H-NMR is tested：Dry extract is dissolved in deuterated reagent, is carried out1H-NMR is tested, and is obtained1H-NMR
Spectrogram；
Step 4, data processing：Will1H-NMR spectrum phase calibration, adjustment baseline, and it is null drift to adjust interior target peak, according to
Characteristic peak determines specific metabolin；
Step 5, using R language pair1H-NMR spectrum carries out orthogonal signalling correction-PLS discriminant analysis, filters out group
Body-sensing should suppress the metabolin of differential expression between group and control group, and the P values that selection is associated with bacterial community induction system are small
The metabolin changed more than 1.1 or less than 0.9 in 0.05 and multiplying power carries out metabolic pathway analysis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109390036A (en) * | 2018-10-31 | 2019-02-26 | 湘潭大学 | A method of excavating selection microalgae grease anabolism marker |
| CN112485283A (en) * | 2020-11-20 | 2021-03-12 | 中国农业科学院农产品加工研究所 | Drying technology evaluation method based on cell membrane integrity change |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109390036A (en) * | 2018-10-31 | 2019-02-26 | 湘潭大学 | A method of excavating selection microalgae grease anabolism marker |
| CN109390036B (en) * | 2018-10-31 | 2021-08-24 | 湘潭大学 | Method for mining and selecting microalgae oil anabolic markers |
| CN112485283A (en) * | 2020-11-20 | 2021-03-12 | 中国农业科学院农产品加工研究所 | Drying technology evaluation method based on cell membrane integrity change |
| CN112485283B (en) * | 2020-11-20 | 2024-05-17 | 中国农业科学院农产品加工研究所 | Drying technology evaluation method based on cell membrane integrity change |
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