Patent Application
20230010612
The present invention belongs to the field of microbiology, and in particular, relates to use of citronellol in preparing a preparation for promoting an expression of a virulence gene toxA of Pseudomonas aeruginosa.
Pseudomonas aeruginosa often induces cystic fibrosis (Cr), meningitis, abscesses, soft tissue infections, urinary tract infections, corneal infections, ventilator-associated pneumonia, and catheter-related infections, Infections induced thereby are highly likely to occur in convalescent patients with low immunity, leading to postoperative wound infections. Acute infections with Pseudomonas aeruginosa can be treated with antibiotics, but when Pseudomonas aeruginosa successfully colonizes in a host, the acute infections would easily turn into chronic infections, showing a permanent biofilm form to result in worsening of organ infections in the host. It is difficult to eradicate Pseudomonas aeruginosa completely with a traditional antibacterial therapy.
The pathogenicity of Pseudomonas aeruginosa depends on the degree of damage to the host by its virulence factor. An exotoxin A of Pseudomonas aeruginosa has a protein structure mainly including three regions, which show different functions in inhibition of protein synthesis after a toxin enters a cell. After the structure and function of the exotoxin A of Pseudomonas aeruginosa have been clearly understood, some scientists have begun to modify and utilize the exotoxin A by genetic engineering, making it medically useful and applicable to clinical medicine.
At present, the exotoxin A of Pseudomonas aeruginosa has been medically used as follows: an antibody is bonded to the exotoxin A to create an immunotoxin, which, with the antibody as a mediator, enters certain specific cells to inhibit protein synthesis in these cells, thereby achieving the purpose of destroying the growth of these specific cells, in particular in the research of anti-cancer drugs; the exotoxin A is used to develop a Pseudomonas aeruginosa vaccine, which is a good vaccine protein that can be applied to vaccine research and development regardless of being prepared into a non-toxic exotoxin-A fragment or fused with other antigens; and in respect of the treatment of liver cancer cells, the exotoxin A has a special function of carrying proteins or DNAs into cells, whereby excellent gene therapy tools or protein therapy drugs can be developed.
Citronellol is a monoterpene with the molecular formula C10H20O. It appears as a colorless liquid with a sweet rose fragrance. Dextro- or laevo-citronellol and racemates thereof can be found in natural plant essential oils. Dextro-citronellol is mainly found in rue oil, citronella oil, and lemon eucalyptus oil; and laevo-citronellol is mainly found in rose oil and essential oils of Pelargonium plants.
An object of the present invention is to provide use of citronellol in preparing a preparation for promoting an expression of a virulence gene toxA of Pseudomonas aeruginosa.
Based on experiments, the present invention demonstrates that citronellol slightly inhibits the growth of Pseudomonas aeruginosa PAO1 strains, and can promote the transcription of Pseudomonas aeruginosa toxA, which can increase the yield of an exotoxin A, namely, an encoded product of toxA.
Therefore, the object of the present invention is to provide the use of the citronellol in preparing the preparation for promoting the expression of the virulence gene toxA of Pseudomonas aeruginosa.
The Pseudomonas aeruginosa is preferably Pseudomonas aeruginosa PAO1.
Preferably, the use of the citronellol in preparing a preparation for promoting the production of an exotoxin A in Pseudomonas aeruginosa is provided.
Preferably, the citronellol is β-citronellol.
Preferably, a concentration of the citronellol in a Pseudomonas aeruginosa culture solution is 0.313-2.5 μL/mL, and further preferably 1.25 μL/mL.
It is found in the present invention that the citronellol at the concentration of 0.313-2.5 μL/mL slightly inhibits the growth of Pseudomonas aeruginosa PAO1 strains, and can promote the transcription of Pseudomonas aeruginosa toxA, which can increase the yield of an exotoxin A, namely an encoded product of toxA. Therefore, the citronellol is applicable to the promotion of the transcription of the Pseudomonas aeruginosa toxA, so as to improve the yield of the exotoxin A.
The embodiments below are intended to further explain the present invention, instead of limiting the present invention.
Preparation of PAO1[(Pseudomonas aeruginosa) PAO1] bacterial suspension: a culture solution of Pseudomonas aeruginosa PAO1 in an exponential growth phase was sampled, centrifuged, washed once with a PBS buffer, resuspended in PBS, and diluted to 108 CFU/mL to obtain the PAO1 bacterial suspension.
1. Experiment on Effects of Citronellol on the Growth of Pseudomonas aeruginosa PAO1
An LB culture medium and citronellol (ii-citronellol) were added to test tubes respectively, and the PAO1 bacterial suspension in the exponential growth phase was inoculated to reach a total volume of 10 mL respectively. The bacterial concentration of PAO1 was each 106 CFU/mL, and the concentration of citronellol was 0 (control), 0.313 μL/mL, 0.625 μL/mL, 1.25 μL/mL, and 2.5 μL/mL, respectively. Samples from several test groups were added to a honeycomb culture plate dedicated to an automatic growth curve analyzer (Bioscreen C), and 350 μL of culture solution was added to each well, with three parallel for each test group. The honeycomb culture plate was placed in the automatic growth curve analyzer, and cultured over shaking at 37° C. for 3 days, and OD600 was measured every hour. Taking OD600 as the ordinate and a culture time as the abscissa, the growth curve of PAO1 under the action of citronellol was drawn to study the effect of the citronellol on the growth of PAO1. The results were shown as in
2. Experiment on Effects of Citronellol on the Expression of Key Genes in a Quorum-Sensing System of Pseudomonas aeruginosa and Virulence Genes Regulated Thereby
The PAO1 bacterial suspension in a logarithmic growth phase was added to 50 mL of sterile LB liquid culture medium to reach a final concentration of 106 CFU/mL for the bacterial solution. Citronellol (1$-citronellol) was added to reach the final concentrations of 0 (three biological replicates in the control group were named A1, A2, and A3, respectively) and 1.25 μL/mL (three biological replicates in the test groups were named B1, B2, B3). Each group was cultured at 37° C. and 180 rpm for 5 h, and centrifuged to collect bacteria, which were then quick-frozen at −80° C. for later use.
Total RNAs were extracted from the bacteria by a Trizol (Thermo Fisher Scientific) kit. After extraction, the purity of the RNAs were detected with an ultra-micro spectrophotometer (Implen, Munich, Germany). An A260/A280 value of each RNA sample should be between 1.8 and 2.0. Reverse transcription and real-time fluorescent quantitative PCR amplification were carried out by using a PrimeScript RT Master Mix kit (Takara, Dalian, China) and an ETC 811 PCR instrument (Eastwin Life Sciences, Inc.). A q-PCR reaction system included Takara SYBR Premix Ex TaqII (Tli RNaseH Plus) (Code No. RR820A), and PCR procedures included: pre-denaturation at 95° C. for 30 s; and denaturation at 95° C. for 5 s and annealing at 60° C. for 34 s, 40 cycles. Based on 10 gene sequences published on the website of GenBank, primers for q-PCR were designed by using software Primer Premier 5.0, and at the same time, a 168 rRNA gene was used as an internal reference gene. The primer sequence parameters are shown in Table 1.
Experimental Results:
The results on the growth curves of Pseudomonas aeruginosa PAO1 under the action of citronellol at different concentrations are shown in
In summary, the experimental results demonstrate that the citronellol slightly inhibits the growth of Pseudomonas aeruginosa PAO1 strains (
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110435173.6 | Apr 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/126812 | 10/27/2021 | WO |
