The Garlic Extracts Block the Quorum Sensing-Controlled Virulence Factor Production in Pseudomonas Aeruginosa
|School||Huazhong University of Science and Technology|
|Keywords||Signaling molecules Garlic extract QS Virulence factors Competitive binding Pseudomonas aeruginosa Mutant Standard strain Bacterial resistance Receptor protein Exotoxin Pyocyanin Regulation of gene expression RNA Gram-negative bacteria Mutant strains Anti-infection treatment Homoserine lactone Receptor binding Pathogens|
Pseudomonas aeruginosa (PA) is a opportunistic pathogen that generally exists in the air, water, skin and intestinal tract of people, especially the most frequently causes nosocomial infection. For to treat, Extensive use of wide-spectrum antibiotics and immunodepressant in recent years has increased the drug-resistance rate and spectrum, multi-drug resistant Pseudomonas aeruginosa(MDRP) is continuously increasing, making clinical treatment difficult. So it also is the pathogenic bacteria in the hospital. It makes people to seek for new anti-infection methods.Recent study showed many bacterial which contain the Gram-positive bacteria and the Gram-negative bacteria rely on quorum sensing (QS) circuits as central regulators of virulence expression. The research that bacterial monitors the quantity change of itself or other bacterium in the surroundings according to concentration of special signal molecules. They adjust related genes to respond to such changes when signal shows concentration has reached certain threshold value. This is called Quorum-sensing system (QS system). The study showed that QS-regulated gene expression contributes to the formation and maintenance of biofilms and their tolerance to conventional antimicrobials and the host innate immune system in Pseudomonas aeruginosa. The Quorum-sensing system of PA has there part of cell-to-cell signaling system and research become more and more popular. The first is LuxR-LuxI which can transcription factor of elastase gene and other various virulence factors of PA. Such as lasA、lasB、aprA、Rpos、xcp. The second is RhlR-RhlI. Deepening research this system is controlled by multiple genes and most of the genes are relevant to bacterial virulence. It control the rhlAB、lasB、aprA、rpos、xcp and so on. At the end, Quinolone signal cascade system was found too. This system which is controlled by QS system can induce lasB to transcribe. The three cell-to-cell signaling system produces a number of quorum-sensing signal molecules (QSSM).The AHL [N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL)] are major signal molecules. These molecules combined the reactivator protein to accommodate the various virulence factors.The garlic is antiauxin that grows in the land with natural broad-spectrum antimicrobial characters. Domestic and foreign studies and reports in the inhibitory aspects of the garlic are many. The first, Garlic has beneficial effects on coagulation, vasodilatation, and serum lipid concentrations. The second, Garlic can inhibit the tumor cell proliferation such as gastric cancer, breast cancer, liver cancer and so on. The third, the garlic has inhibiting and killing function to many kinds of bacteria, fungi and protozoon. It has been testified to be a kind of green natural biological antiseptic with safety, high-efficiency nature characters and so on. The garlic could inhibit the growth of spoilage bacteria and pathogenics. Recently, QS-blocking properties of garlic have been research. The studies were described:Extraction of garlicPreparation of garlic extract, The garlic extract used in all the experiments. Prior to extraction, the stem and dead leaves were removed from the garlic bulbs and washed them. A total of 150 g of garlic cloves was minced with a standard kitchen blender along with toluene. After the sterile water was added, and the mixture was stirred at room temperature, after which the two phases were allowed to form. The water phase was separated from the organic phase, sterile filtered, and used as raw extract in the experiment.The growth curve of PAThe single colonies of PAO1 strain and PAO-JP2 strain were selected and picked, and then put in 5ml culture fluid respectively under the condition of shaking cultivation at 37℃. According to the bacterial counting of 6h、12h、18h and 24h, draw the growth curve.Reverse transcriptase PCR (RT-PCR)The single colonies of PAO1 strain and PAO-JP2 strain were selected and picked, and then put in 5ml culture fluid respectively and cultivated for a night under the condition of shaking cultivation at 37℃. According to the reagent instruction to abstraction the RNA, The total RNA quality and concentration were checked with RNA meter and then the Random primers were applied to reversely transcribe RNA into cDNA according to reagent instruction. cDNA of 2 bacterial strains mentioned above was taken as templates, and then conducted extension method of lasB, toxA, aprA and 16s rRNA respectively. HMIAS-2000 High-resolution color figure analysis system was used to compare the three genes and internal references.Test activity of exotoxin A through NAD (Co I) methodActivity of exotoxin A was tested with NAD relied develop system. Comparison of positive and negative solutions was included in tests. Same amount of NAD was contained in positive tubes but no toxin, while negative tubes, no NAD. All other conditions were the same with the above-mentioned testing tubes, 0 value of OD490 colorimetric estimation was adjusted according to negative tubes, and develop suppression rate was calculated. Statistic analysis was conducted based on 6 parallel tests.Test of ProteaseThe single colonies of PAO1 strain and PAO-JP2 strain were selected and picked, and then put in 5ml PTSB culture fluid respectively and cultivated for a night under the condition of shaking cultivation at 37℃until the OD540 2.5~2.6. After centrifuge, the culture bacterial supernatant were added to 900μl of ECR buffer containing Elastin Congo red, and then incubated at 37°C. The absorption of the supernatant was measured at 495 nm. Statistic analysis was conducted based on 6 parallel tests.Test of Pyocyanin The single colonies of PAO1 strain and PAO-JP2 strain were selected and picked, and then put in 5ml PB culture fluid respectively and cultivated for a night under the condition of shaking cultivation at 37℃until the OD540 2.5~2.6. Five milliliters of PB culture was extracted in chloroform. The chloroform phase was transferred to a fresh tube and mixed with HCl, and the mixture was gently shaken to bring the pyocyanin to a pink phase. After centrifugation, the absorbance of the supernatant was removed and measured at 520nm. Statistic analysis was conducted based on 6 parallel tests.Conclusions:In this research the garlic extract can suppression to elastinase, pyocyanine and exotoxin A. In conclusion, the QS system can control the virulence of PA. In this research, virulence factors of PA were depressed obviously by the garlic extract. It indicated that there are compounds which can conduct and actions the Quorum sensing inhibitors in the garlic extract. They can interfer QS system for cut down virulence of PA, if this idea can be used in clinical will be fine. In particular, the monomer compound which comes from the garlic extract as a new antibacterial will offer a new way for solving drug resistance.