CN111166732A - Application of diallyl disulfide in preparation of drug for inhibiting pseudomonas aeruginosa two-component system - Google Patents
Application of diallyl disulfide in preparation of drug for inhibiting pseudomonas aeruginosa two-component system Download PDFInfo
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- CN111166732A CN111166732A CN202010034758.2A CN202010034758A CN111166732A CN 111166732 A CN111166732 A CN 111166732A CN 202010034758 A CN202010034758 A CN 202010034758A CN 111166732 A CN111166732 A CN 111166732A
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/095—Sulfur, selenium, or tellurium compounds, e.g. thiols
- A61K31/10—Sulfides; Sulfoxides; Sulfones
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Abstract
The invention discloses application of diallyl disulfide in preparation of a pseudomonas aeruginosa two-component system inhibiting drug. The invention finds that the DADS can not generate obvious inhibition effect on the growth of the pseudomonas aeruginosa PAO1, but can obviously inhibit a two-component system of the pseudomonas aeruginosa PAO1, has the activity of inhibiting the virulence factor of the pseudomonas aeruginosa, and therefore, can be used for preparing the medicine for inhibiting the pseudomonas aeruginosa virulence factor.
Description
The technical field is as follows:
the invention belongs to the technical field of microorganisms, and particularly relates to application of diallyl disulfide in a pseudomonas aeruginosa two-component system.
Background art:
two-component systems, also known as two-component signal transduction systems, enable bacteria to sense, respond and adapt to environmental or intracellular changes. The two-component system is responsible for environmental information processing and signal transduction and simultaneously regulates and controls the expression of some virulence genes and virulence factors of bacteria. Garlic is a common food, which has been used as a folk medicine for thousands of years. Modern scientific research proves that the sulfur-containing compounds in the garlic essential oil have strong antibacterial and anti-inflammatory effects and have inhibitory effects on various bacteria, fungi and viruses. Diallyl disulfide (DADS) is a sulfur-rich compound in garlic essential oil.
The invention content is as follows:
the invention aims to provide application of diallyl disulfide in preparation of a drug for inhibiting a pseudomonas aeruginosa two-component system.
Experiments show that the DADS can not generate obvious inhibition effect on the growth of the pseudomonas aeruginosa PAO1, but can obviously inhibit a two-component system of the pseudomonas aeruginosa PAO1, and has the activity of inhibiting virulence factors.
Therefore, the invention provides the application of diallyl disulfide in preparing a drug for inhibiting a two-component system of pseudomonas aeruginosa.
The invention finds that the DADS can not generate obvious inhibition effect on the growth of the pseudomonas aeruginosa PAO1, but can obviously inhibit a two-component system of the pseudomonas aeruginosa PAO1, has the activity of inhibiting the virulence factor of the pseudomonas aeruginosa, and therefore, can be used for preparing the medicine for inhibiting the pseudomonas aeruginosa virulence factor.
Description of the drawings:
FIG. 1 is a KEGG pathway map of a Pseudomonas aeruginosa two-component system under the action of DADS and differential expression of its genes.
FIG. 2 is a KEGG pathway map of a Pseudomonas aeruginosa two-component system under the action of DADS and differential expression of its proteins.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
PAO1 bacterium [ Pseudomonas aeruginosa (Pseudomonas aeruginosa)]Preparation of suspension: PAO1 culture fluid in exponential growth phase was sampled, centrifuged, washed once with PBS buffer, resuspended in PBS, and the bacterial concentration was diluted to 108CFU/mL。
1. Analysis of the Effect of DADS on the P.aeruginosa PAO1 transcriptome by high-throughput RNA sequencing technology
Growing the mixture in exponential phase at a concentration of 108CFU/mL of P.aeruginosa PAO1 was inoculated onto 50mL of LB medium, followed by the addition of DADS at a concentration of 0 (control) or 0.64mg/mL, and the experiments were set up in triplicate. All 6 experimental groups were incubated in a 37 ℃ water bath incubator at 180rpm for 5 h. The cells from the three control groups and the three DADS-treated groups were sampled and centrifuged, and then the cell pellets were snap frozen at-80 ℃.
1.1RNA sample preparation
Total RNA was isolated from cell samples using TRIZOL reagent (seimer feishell scientific, usa) according to the manufacturer's protocol. RNA purity was determined using a nanophotometer (IMPLEN, Calif., USA) with an A260: A280 ratio of 1.8 to 2.0 for all RNA samples. RNA concentration was measured using a Qubit 2.0 fluorescence quantifier in combination with an RNA quantitative analysis kit (Life technologies, USA, Calif.). Agilent 2100 biochip Assay system and its Kit RNArano 6000Assay Kit for RNA sample integrity detection (Agilent technologies, Inc., Calif., USA); the RNA integrity of all samples was greater than 7.0.
1.2 library construction of specific strands
Transcriptome sequencing
RNA sequencing was performed by the Bioinformation technology, Inc., of kindergarten (Beijing, China). Each sample required 3ug of RNA as starting material for RNA sample preparation. Sequencing libraries were generated using the NEBNext super-targeted RNA library prep kit from Illumina (new britain biotechnology limited, usa) according to the manufacturer's recommendations and adding an index code to the attribute sequence of each sample. Briefly, mRNA was purified from total RNA using poly-T oligonucleotide magnetic beads. rRNA was isolated from mRNA using a special kit. And disrupted with divalent cations at high temperature in NEBNext first strand synthesis reaction buffer (5X). First strand cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNaseH). Subsequently, the second strand of cDNA is synthesized with DNA polymerase I and RNase H. Phusion high-fidelity DNA polymerase, PCR universal primers and Index primers are adopted for PCR amplification. Finally, the product was purified (nucleic acid purification system) and the quality of the sample pool was assessed using the Agilent 2100 biochip assay system. RNA sequencing was performed on the Illumina sequencing platform by Beijing Nuo He Sourceology science and technology, Inc.
Reading reference genomic maps, novel gene and gene structure analysis, quantification of gene expression levels
Reference genome and gene model annotation files directly from NCBI1And downloading a genome website. A reference genome index was constructed using bowtie2-2.2.3 and clean reads aligned to the reference genome. Rockhopper was used to identify novel genes, operons and transcription start sites. The system can efficiently and accurately analyze the bacterial RNA sequence data, thereby assisting in analyzing the bacterial transcriptome. Then, a Time Delay Neural Network (TDNN) is used for extracting 700 base pairs upstream of the transcription initiation site to predict the promoter. HTSeq v0.6.1 was used to calculate the number of reads per gene, and the number of reads per kilobase corresponding to an exon per million Fragments (FPKM) was calculated based on the length of the gene and the reads count of the gene.
1https://www.ncbi.nlm.nih.gov/nuccore/AE004091.2
Differential expression analysis of differential expression genes, GO enrichment analysis and KEGG pathway analysis
Differential expression analysis between groups was performed using the DESeq R package (1.18.0), and the resulting p-values were adjusted by controlling the false discovery rate using the methods of Benjamini and Hochberg. Corrected p-value of the Gene by DESeq identification<0.05, it is differentially expressed. And carrying out Gene Ontology (GO) function enrichment analysis on the differentially expressed genes by using the GOSeq R packet, and correcting the length deviation of the genes. GO terminology correcting p-values<0.05 was considered to be significantly enriched for differentially expressed genes. Kyoto gene and genome encyclopediaThe book KEGG database is a resource library for understanding various biological system functions and attributes from molecular level information, and particularly refers to a large-scale molecular data set generated by high-throughput experimental techniques such as genome sequencing2. Differential expressed gene KEGG pathway enrichment analysis was performed with KOBAS software.2http://www.genome.jp/kegg/
1.3 registration number
The RNA sequence dataset can be found in the NCBI gene expression integrated database (GEO), search number GSE 118801.
Analysis of the Effect of DADS on Pseudomonas aeruginosa PAO1 proteome by iTRAQ high throughput sequencing technology
To investigate the effect of DADS on the Pseudomonas aeruginosa proteome, a concentration of 10 was used8CFU/mL of P.aeruginosa PAO1 in exponential growth phase was inoculated into 50mL of LB medium sample. DADS was then added at concentrations of 0 (control) or 0.64mg/mL and the experiments were set up in triplicate. All 6 experimental groups were incubated in a 37 ℃ water bath incubator at 180rpm for 5 h. The cells from the three control groups and the three DADS-treated groups were sampled and centrifuged, and then the cell pellet from each experimental group was snap frozen at-80 ℃.
2.1 sample preparation and iTRAQ labeling
Obtaining protein extract by RIPA lysate. Protein concentrations were determined by the Bradford method using BSA as standard (BCA kit, biotechnology) according to the manufacturer's protocol. iTRAQ protein sequencing analysis was performed by guangzhou hujun biotechnology limited (guangzhou, china). The protease cleavage was performed by the FASP method (Wisnewski et al, 2009; Lan et al, 2012; Kambiranda et al, 2014). According to the user's manual, iTRAQ labeling was performed using iTRAQ reagents-8 plex Kit (AB SCIEX, USA). Samples of pseudomonas aeruginosa PAO1 from the three control groups were labeled with iTRAQ as 114, 115 and 116, respectively, while samples of pseudomonas aeruginosa PAO1 from the three DADS-treated groups were labeled with iTRAQ as 117, 118 and 119, respectively.
2.2 high pH reverse phase chromatography
The iTRAQ-labeled sample was diluted to 100uL with 20mmol/L of HCOONH4 and 2mol/L of NaOH (pH 10) and then used for high performance liquid chromatography analysis. 20mmol/L HCOONH4, 2mol/L NaOH and 80% CAN are used as mobile phase B, 5% to 40% gradient elution is carried out for 30min, the flow rate is 0.2mL/min, and the polypeptide is separated. The UV detector was set at 214/280nm and fractions were collected every 1 minute. A total of 10 fractions were collected for each sample and dried using a vacuum centrifuge.
2.3 reversed phase liquid chromatography-Mass Spectrometry coupled analysis
The polypeptide was acidified with 50% CF3COOH and then eluted with a mobile phase B gradient of 5% to 40% for 70min at a flow rate of 300 mL/min. In the information-dependent mode, mass spectrometry was performed using TripleTOF 5600+ mass spectrometry system (AB SCIEX, usa). In the high resolution mode, MS spectra were obtained over a mass-to-charge ratio range of 400-1250 with an integration time of 250MS per spectrum. In one cycle, 50 precursors per MS spectrum were selected for fragmentation at the maximum, with a minimum cumulative time of 100MS and a dynamic exclusion time of 20s per precursor. Tandem mass spectrometry records data in a high sensitivity mode under the conditioning of rolling collision energy and iTRAQ reagent collision energy.
2.4 data analysis
Protein quantification and identification analysis was performed using the protein pilot software (version 4.0, revision 148085, applied biosystems, usa) using the Paragon algorithm as a search engine. The prescribed processing includes quantization, offset correction, and background correction. All identified proteins had confidence levels of 95% or more, and the protein confidence threshold cutoff was set at 1.3 (not used), with at least one polypeptide having a confidence level of greater than 95%. To describe the significance of differential expression of proteins, fold differences were measured>2 or<0.5 is set as the cutoff value. At QuickGO website3(Note blacklist)4) GO annotation and enrichment analysis were performed on differentially expressed proteins. The GO project classifies proteins into three categories, biological processes, cellular composition and molecular function, according to protein annotation. After annotation was complete, the corresponding enzyme coding sequences for each protein were reflected in the known metabolic pathways of KEGG2 for biological pathway analysis.
3http://www.ebi.ac.uk/QuickGO/Dataset.html
4http://www.ebi.ac.uk/QuickGO/AnnotationBlacklist.html
2.5 registration number
Proteomic mass spectral data has been stored via the ide repository to the proteomxchange database under the accession number PXD 011144.
The experimental results are as follows:
transcriptome sequencing results showed that a number of P.aeruginosa PAO1 genes were differentially expressed following DADS treatment. We found that DADS treatment differentially expressed more than 50% of the genes (3374 out of 5630 genes) by more than 2-fold over all the genes detected. 1725 genes are down-regulated by more than 2 times, and 1649 genes are up-regulated by more than 2 times. To further investigate the biological function of differentially expressed genes, the function of the identified genes was classified using KEGG analysis. Data enrichment analysis shows that after DADS treatment, the PAO1 strain has significant enrichment of differentially expressed genes in a two-component system, as shown in FIG. 1, red indicates that the transcription level is up-regulated, and green indicates that the transcription level is down-regulated, and the graph shows that the genes with down-regulated expression are mainly used as the main genes.
Proteomic sequencing results showed that DADS treatment differentially expressed more than 2-fold over 30% of the proteins detected (1133 out of 3304 proteins). Following DADS treatment, 425 protein expression was downregulated > 2-fold and 708 protein expression was upregulated > 2-fold. The KEGG analysis result also shows that the differentially expressed proteins are significantly enriched in the two-component system pathway of the PAO1 strain, as shown in fig. 2, red indicates proteins with up-regulated protein expression level, and green indicates proteins with down-regulated protein expression level, and it can be seen from the figure that proteins with down-regulated protein expression level are mainly used.
And combining the analysis data of the transcriptome and the proteome, wherein the differentially expressed genes/proteins of the transcriptome and the proteome are obviously enriched in a KEGG passage of a two-component system, and a plurality of the differentially expressed genes/proteins are related to the expression regulation of the virulence factors of the pseudomonas aeruginosa. Therefore, DADS can inhibit the gene and protein expression of a two-component system of pseudomonas aeruginosa, and further inhibit the production of virulence factors of the bacterium.
Claims (1)
1. Application of diallyl disulfide in preparation of medicines for inhibiting pseudomonas aeruginosa two-component system.
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Citations (3)
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| CN101385737A (en) * | 2008-10-31 | 2009-03-18 | 西北大学 | Antibiotic effective ingredient and use thereof |
| CN103328441A (en) * | 2010-12-08 | 2013-09-25 | 丹麦技术大学 | Process for the manufacture of ajoene derivatives |
| CN106361732A (en) * | 2016-08-26 | 2017-02-01 | 广东省微生物研究所 | Application of diallyl disulfide in preparing a pseudomonas aeruginosa quorum sensing quenching agent |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101385737A (en) * | 2008-10-31 | 2009-03-18 | 西北大学 | Antibiotic effective ingredient and use thereof |
| CN103328441A (en) * | 2010-12-08 | 2013-09-25 | 丹麦技术大学 | Process for the manufacture of ajoene derivatives |
| US20140303070A1 (en) * | 2010-12-08 | 2014-10-09 | Danmarks Tekniske Universitet | Process for the manufacture of ajoene derivatives |
| CN106361732A (en) * | 2016-08-26 | 2017-02-01 | 广东省微生物研究所 | Application of diallyl disulfide in preparing a pseudomonas aeruginosa quorum sensing quenching agent |
Non-Patent Citations (4)
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