LU501586B1 - Application of a Trichodermin - Google Patents
Application of a Trichodermin Download PDFInfo
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- LU501586B1 LU501586B1 LU501586A LU501586A LU501586B1 LU 501586 B1 LU501586 B1 LU 501586B1 LU 501586 A LU501586 A LU 501586A LU 501586 A LU501586 A LU 501586A LU 501586 B1 LU501586 B1 LU 501586B1
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- trichodermin
- trichophyton mentagrophytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P9/00—Drugs for disorders of the cardiovascular system
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Abstract
The present invention discloses the application of a trichodermin used for preparation of drugs for treating skin diseases caused by Trichophyton mentagrophytes. The trichodermin in the present invention is from yew Trichoderma strain ZJUF0986. It can inhibit Trichophyton mentagrophytes, has the potential of serving as the drug of treating skin diseases caused by Trichophyton mentagrophytes and provides a scientific basis for treating with biological prevention and control methods the fungal skin diseases of livestocks caused by Trichophyton mentagrophytes. The in vitro bacteriostasis results show that Trichophyton mentagrophytes colony growth is totally inhibited under the action of trichodermin 1000 μg/mL.
Description
a . . LU501586
Application of a Trichodermin
The present invention relates to the application of a metabolite for biological prevention of fungi, particularly to application of a trichodermin.
Trichophyton mentagrophytes (Tm) 1s an important zoonotic skin tinea. It mainly infects the skin corneum and affiliated keratinization tissues of humans and animals, doing harm to the health of humans and animals. Trichophyton mentagrophytes skin diseases are mainly treated with drugs. Currently, there are multiples clinical drugs.
However, the overall efficacy of drugs is instable, and diseases are likely to relapse due to the drug tolerance arising from long-term treatment and repeated administration. Hence, it is urgent to seek for a novel, efficient and long-acting drug.
To finding new compounds from natural products is an important approach of developing new drugs. Microorganisms are a major source of natural products in nature. A variety of fungi in the Trichoderma can be used for biological prevention and control of plant diseases. The biological control mechanisms include hyperparasitism, antibiosis, competition, etc. The antibiotic substances produced by them play an important role in the biological control mechanism. The separation identification and function research of endophytic fungi is an emerging research field.
Previous research has revealed that endophytic fungus Trichoderma strain
ZJUF0986 and the active substance trichodermin from this strain are active in inhibiting plant pathogenic fungi.
0; LU501586
The present invention aims to provide the application of a trichodermin that can inhibit Trichophyton mentagrophytes, provide a scientific basis for treating with biological prevention and control methods the skin diseases of domestic rabbits caused by Trichophyton mentagrophytes, and serve as a drug for treating the skin diseases caused by Trichophyton mentagrophytes.
To achieve the above purpose, the present invention discloses the application of a trichodermin used for preparation of drugs for treating skin diseases caused by
Trichophyton mentagrophytes.
Preferentially, said trichodermin is an active metabolite obtained from
Trichoderma strain ZJUF0986.
Preferentially, said trichodermin inhibits the colony growth of Trichophyton mentagrophytes.
Preferentially, the concentration of said trichodermin is greater than or equal to pg/mL and smaller than 20 mg/mL.
Preferentially, the concentration of said trichodermin is greater than or equal to 10 pg/mL, and its inhibition rate for the colony growth of Trichophyton mentagrophytes is equal to or greater than 18%.
Preferentially, the concentration of said trichodermin is greater than or equal to 100 pg/mL, and its inhibition rate for the colony growth of Trichophyton mentagrophytes is equal to or greater than 40%.
Preferentially, the concentration of said trichodermin is greater than or equal to 1000 pg/mL, and it totally inhibits the colony growth of Trichophyton mentagrophytes.
Preferentially, said trichodermin inhibits the spore germination of Trichophyton mentagrophytes.
Preferentially, the concentration of said trichodermin is greater than or equal to 0100 ng/mL, and its inhibition rate for the spore germination of Trichophyton mentagrophytes 1s equal to or greater than 69%.
Preferentially, the concentration of said trichodermin 1s greater than or equal to 100 ug/mL, and its inhibition rate for the spore germination of Trichophyton mentagrophytes 1s equal to or greater than 91%.
The present invention also aims to provide a method for regulating and controlling the function of trichodermin for Trichophyton mentagrophytes. This method comprises the following contents: By lowering the expression of gene CDR 1 or knocking out the gene CDRI, the function of trichodermin for inhibiting
Trichophyton mentagrophytes is enhanced.
The application of trichodermin in the present invention has the following advantages:
The trichodermin in the present invention is obtained through separation and purification of Trichoderma strain ZJUF0986. It can inhibit Trichophyton mentagrophytes, provide a scientific basis for treating with biological prevention and control methods the skin diseases of domestic rabbits caused by Trichophyton mentagrophytes, and serve as a drug for treating the skin diseases caused by
Trichophyton mentagrophytes.
The in vitro bacteriostasis results show that Trichophyton mentagrophytes colony growth is totally inhibited under the action of trichodermin 1000 pg/mL, indicating that trichodermin 1000 pg/mL can totally inhibit mycelial growth of
Trichophyton mentagrophytes. Under the action of the trichodermin, relevant expression genes of mitochondria declined significantly, indicating that mitochondria were damaged in the trichodermin processing process, or participated in the tolerance process of trichodermin. Several ABC genes of Trichophyton mentagrophytes declined under the action of trichodermin. By knocking out one
ABC gene CDR], the drug sensitivity testing result shows that gene CDR1 plays a key role in Trichophyton mentagrophytes' tolerance of trichodermin.
Figure 1 shows the Tm colony growth under the action of different concentrations of trichodermin in experimental case 1 of the present invention.
Figure 2 shows the Tm colony diameter under the action of different concentrations of trichodermin in experimental case 1 of the present invention.
Figure 3 shows the Tm sporulation quantity under the action of different concentrations of trichodermin in experimental case 1 of the present invention.
Figure 4 shows the Tm spore germination rate under the action of different concentrations of trichodermin in experimental case 1 of the present invention.
Figure 5 shows the Tm ultrastructure under the action of different concentrations of trichodermin in experimental case 1 of the present invention.
Figure 6 shows the Tm mitochondria dyeing observation result under the action of different concentrations of trichodermin in experimental case 1 of the present invention.
Figure 7 shows the interaction between trichodermin and Tm in experimental case 1 of the present invention.
Figure 8 shows the observation result of interaction between the GFP transformant of trichodermin and the RFP transformant of Tm in the experimental case 1 of the present invention.
Figure 9 shows the drug sensitivity testing result of strains with gene CDR1
5 LU501586 knocked out for trichodermin in the experimental case 2 of the present invention.
Figure 10 shows the treatment result of the rabbit skin disease caused by trichodermin in the experimental case 3 of the present invention.
Next, clear and complete descriptions of the technical solution in the embodiments of the present invention are stated; Obviously, the described embodiments are only some parts, not the whole ones. Based on the embodiments of the present invention, all the other embodiments obtained by those ordinarily skilled in the art without making creative endeavors fall into the scope of protection of the present invention.
The inhibition function of trichodermin for Tm in the experimental case 1 1. Colony growth and sporulation quantity of Tm under the action of trichodermin
The trichodermin is an active metabolite from Trichoderma strain ZJUF0986 (the same as that in the following experimental case), tested strains used are
Trichophyton mentagrophytes, the drug sensitivity plates are SDA plates containing 10, 100, 1000 LU g/mL trichodermin respectively, and three repeated SDA plates for each concentration; The cake punching method was used for making Tm cakes for culture on the drug sensitivity plates (the same as that in the following experimental cases), culture was conducted for 6 days, the culture was observed and photographed, and the colony size was measured; The colonies were washed with saline, filtered with two layers of filter paper, and spores were counted with a hemocytometer to calculate the sporulation quantity per SDA plate.
Figure 1 shows the Tm colony growth under the action of different concentrations of trichodermin in experimental case 1 of the present invention; 0100
Figure 2 shows the Tm colony size under the action of different concentrations of trichodermin in experimental case 1 of the present invention; After growing for 6 days, as the concentration of trichodermin rose, the Tm colonies on the drug sensitivity plates decreased gradually, and were significantly smaller than colonies on the SDA (CK) of the blank control group (P<0.01); When the concentration of trichodermin was 10, 100 u g/mL, the colony growth inhibition rate was 18% and 40% respectively; When the concentration of trichodermin reached 1000 u g/mL, the Tm colony growth was totally inhibited. Figure 3 shows the Tm sporulation quantity under the action of different concentrations of trichodermin in experimental case 1 of the present invention; The number of spores of each Tm colony on the drug sensitivity plates where the concentration of trichodermin was 10 u g/mL and 100
Wu g/mL was 8.4 X 10” and 2.3 X 107 respectively, which was significantly lower than the 1.84 X 10° of the control group, and there was a dose dependence relationship. 2. The spore germination rate of Tm under the action of trichodermin 5x10°/mL Tm spore solution containing 0, 10, 100 pg/mL trichodermin was prepared, and cultured for 14 h under 280 in a dark environment, the spore germination situation was observed, and the spore germination rate was calculated by virtue of Calcofluor White dyeing. The specific operations are as follows: Spore solution cultured was dripped onto the glass plate, dyed with Calcofluor White dye solution, and observed immediately under the fluorescence microscope, the spore germination situation was observed, and the spore germination rate was calculated.
As shown in Figure 4, it is the Tm spore germination rate under the action of different concentrations of trichodermin in experimental case 1 of the present invention (A for observation under the fluorescence microscope, B for the statistical spore germination rate). After culture for 14h, it was found through observation with the Calcofluor White dyeing that the spore germination rate of Tm processed with u g/mL and 100 u g/mL trichodermin was 13.3% and 3.9% respectively, which 0106 significantly declined compared with the blank control group (43.5%), the spore germination inhibition rate was 69% and 91% respectively (Table 1), and the spore germination rate inhibition degree was correlated with the rise of the concentration of trichodermin.
Table 1 is the data about colony growth, sporulation quantity and spore germination inhibition rate
SDA (CK) Trichodermin 10 Trichodermin 100 pg/mL pg/mL
Colony diameter (cm) 4.73 3.9 2.85
Colony growth inhibition rate 0 18% 40%
Number of spores (pcs./plate) 1.84x108 8.4x107 2.3x107
Sporulation inhibition rate 0 54% 88%
Spore germination rate 43.5% 13.3% 3.9%
Spore germination inhibition 0 69% 91% rate 3. Tm microexamination under the action of trichodermin
SDA plates containing 10 and 100 pg/mL trichodermin were made, inoculated
Tm was cultivated for 6 days, and the ultrastructure was observed under the transmission electron microscopy. In the meanwhile, Tm was processed with mitochondria dyeing with MitoTracker Green FM green dye, and observation was conducted under the confocal microscopy. MitoTracker Green FM green dye is a bright green fluorescence probe (F488nm) which hardly fluoresces in water solution, and only fluoresces when it gathers in the mitochondrial lipid environment. Before use, 1 mM stock solution was prepared with DMSO and the dye according to the instruction, and then the stock solution was added into 1000 uL water to prepare the dyeing liquid. 15 u L water was dripped onto the glass slide, mycelia were picked
, LU501586 up and placed in water, 15 u L dyeing liquid was added for dyeing, and observation was immediately conducted under the confocal microscopy.
Figure 5 shows the ultrastructure observation result of Tm under the action of different concentrations of trichodermin in experimental case 1 of the present invention. Under the electron microscope, the mitochondria of Tm expanded, and the mycelia form changed; Figure 6 shows the Tm mitochondria fluorescence staining observation result under the action of different concentrations of trichodermin in experimental case 1 of the present invention. Through dyeing with mitochondria dye MitoTracker Green FM, it was found through observation that the quantity of mitochondria in Tm mycelia increased significantly under the action of trichodermin. 4. Interaction between trichodermin and Tm
Trichodermin and Tm were inoculated on an SDA plate by keeping an interval of Scm. After three days of culture, two colonies approached each other. The interaction between them was observed under the microscope.
Figure 7 shows the interaction between trichodermin and Tm in experimental case 1 of the present invention. After three days of culture, trichodermin mycelia extended to and invaded Tm, involved and twisted with Tm mycelia, and Tm retreated under the attack of trichodermin.
Figure 8 shows the observation result of interaction between the GFP transformant of trichodermin and the RFP transformant of Tm in the experimental case 1 of the present invention. Under the confocal microscopy, by virtue of the mark of GFP (green fluorescent protein) and RFP (red fluorescence protein), interaction between trichodermin and Tm and the twisting of mycelia could be seen more obviously.
It was found through analysis on data of the transcriptome group that under the action of trichodermin, the expression of several mitochondria genes (bifunctional 0100 dethiobiotin synthetase/7,8-diamino-pelargonic acid aminotransferase, zinc-binding oxidoreductase, RutC family protein, oxygen-dependent coproporphyrinogen-III oxidase, biotin synthase, arginine biosynthesis bifunctional protein) declined, indicating that mitochondria were damaged in the trichodermin processing process, or participated in the tolerance process of trichodermin.
Experimental Case 2 Impacts of Gene CDR1 on the Drug Sensitivity
Experiment of Trichodermin
Access of gene CDRI lacked strains: By using Tm strain genome DNA as the template, CDR1 upstream segment (SEQ ID NO.1) was amplified for 1.2 kb with
PCR, and CDR1 downstream segment (SEQ ID NO.2) was amplified for 1.1 kb with
PCR, and then these two gene segments were inserted respectively into p1300-KO (introduced into pCAMBIA1300 with hygromycin gene) (Cambia, Canberra,
Australia) to form CDRI1 knock-out carrier pKO-TmCDR1. PKO-TMCDRI was introduced into Tm strains via AtMT, and the transformants were screened with CM plates containing 250ug/mL hygromycin B. CDRI1 lacked strains T9-21 and T4-12 were obtained with multiple-genome PCR amplification method and real-time quantitative PCR screening and verification. The homologous gene of TMCDR1 was introduced into T9-21 lacked strain to obtain the complementary strains PNMNR6 (5) and PNMG7 (6).
Impacts of gene CDR1 on the drug sensitivity experiment of trichodermin: Tm strains, CDRI1 lacked strains T9-21 and T4-12, gene CDRI recovered strains
PNMNR6 (5) and PNMG7 (6) were inoculated on SDA plates containing 10 and 100ug/mL trichodermin for culture. 6 times of repetition was conducted for each time of processing. 6 days of culture was conducted. Figure 9 shows the result of impacts of gene CDRI on the drug sensitivity experiment of trichodermin in experimental case 2 of the present invention. After 6 days of culture, CDRI1 lacked
0 Co LU501586 strains T9-21 and T4-12 were significantly inhibited under the action of 10, 100ug/mL trichodermin compared with wild strains, while the sensitivity of gene
CDRI recovered strains PNMNR6 (5) and PNMG7 (6) to trichodermin reduced.
Experimental Case 3 Animal Skin Toxic Counteraction and Treatment
Experiment
Toxic counteraction was conducted for the rabbit skin with Tm at first. After toxic counteraction, individuals with severe morbidity were used for therapeutic test.
Treatment was conducted by smearing 1000 ng/mL and 20 mg/mL trichodermin once a day (2mL for each time). Treatment was conducted for three consecutive days.
The treatment result was observed and photographed after 14 days of treatment.
Figure 10 shows the animal skin toxic counteraction and treatment experiment result in experimental case 3 in the present invention. After treatment by smearing 1000 pg/mL trichodermin, the Tm skin disease wound healing effect was significantly improved. However the dosage of trichodermin shall not be high, because toxic injury of skin can be caused when the concentration of trichodermin reaches 20 mg/mL.
The content of the present invention has been introduced in detail with the above- mentioned preferential embodiments, but it shall be realized that the above description shall not be regarded as restrictions on the present invention. Multiple kinds of modification and substitution of the present invention by technicians in this field after reading the aforesaid content will be obvious. Hence, the protection cope of the present invention shall be defined by the claims attached.
or LU501586
Sequence Listings <110> Zhejiang Academy of Agricultural Sciences <120> Application of a Trichodermin <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1240 <212> DNA <213> Artificial sequence <400> 1 cagtattgca gagacaaccc ttagcggaag tcctctacag tgctgggaca acagcactag 60 aggccttgac agtgccaatg cecttgaatt tgtcaaatca ctccgcctgt ctactaaata 120 ctcgggaacc actgceccatcg ttgccatcta ccaggccggt caagcaatct acgacatett 180 cgacaaagcg gttgttctat acgagggaca tcagatctac tttggaaatg ctgtccgage 240 aaaagaatac tttatcgaga tgggttttga ctgtcctage cgacagacca ctgctgattt 300 cttaacttcg gtcaccagtc cgtccgaacg gagagtcagg cctggectacg agtctegtgt 360 accccagaca ccggectgagt ttgctcagcg atggaaagaa agtgaagaca gacgaattct 420 catgcaggag attgatgaat acaataagac ctacccgcta catggcgaac agctacagaa 480 atteccaggec tctcggtcag ctgaaaagtc taggtcagec agcaagtect cgecgtatac 540 cctctettac cccatggaga tcaaactttg catgtggegt ggattccaga gactgaaggg 600 agacatgtcc atgactctga cctccataat tggaaacatc gccatgtcce tgatcattge 660 cagtgtgttc tataaccagc aagaaaccac tgacagcttt ttctccegtg gctetctect 720 tttctttgee attctcatga acgectttge tagttccctg gaaattttga ccctetggea 780 LU501586 tcaacggcec atcgtggaaa aacatgataa atacgctcta tatcatccat cctccgaage 840 catcagttct atacttgtcg atatgcctge aaaattggec gtggccateg tgtttaatct 900 gatcatatat tttatgacca acctaaggag aacccccggt cacttetttg tetttttect 960 cttetcette accacgacce tgaccatgte taacgtette cgttccattg ctgecgtgte 1020 tcgtacctta teccaagctt tggtecctac ctcaatettt atgettgeet tggttatcta 1080 cactggtttt accattccag ttcgtgatat gecgaccctgg ttcaagtgga tcagttacat 1140 caaccccatt cagtacgcgt ttgagtctct catgatcaac gagttccatg accgagaatt 1200 taaatgtget gcctatatac ccagtggtce cggatactec 1240 <210> 2
<211> 1091
<212> DNA
<213> Artificial sequence
<400> 2 gactcctcgg ttggccggat acggtgaatt tgectatgecce ctgtggaaac agtatctcat 60 tetccagcac cgtatgttcc agcagtactg gegtagtcet gactatatct attcgaaagce 120 ctgtcttgee attgttcecgg tatgctaaca teccetttec cttggttata ttegtttgaa 180 cactaacata ttcctctaga ctctattcat tggttttact ttctacaagg aacaagtcag 240 tttgcaaggt atccagaacc aaatgttcge cattttcatg tttatgattc tcttccccaa 300 tcttgtccag caaatgatge cctactttgt catccaacga tctctctacg aagtccgega 360 acgaccctca aaaacatact cctggatage atttatgata tcatcagtca ttgttgaaat 420 accctggaat gectctectga ctgtececcgc cttettetge tggtactacc ccattggett 480 ctataagaac gcaatcccaa ccgacgccgt gactgaacga agtggcacca tgttcecttget 540 catcctgatc ttcctcatgt tcagctcgac attcagttec atggttattg ccggtatcga 600 gcaagcggaa acaggtggaa acattgccca getttgette teccecctcacce tegtettetg 660 cggtgttcte gttagtccaa ccgeccatgee cggattectgg atcttcatgt accgettate 720 LU501586 tcecgttcaca tatttcgttt cagccgtgtt atctactgga gtcggaagaa cagatattgt 780 gtgcgetgee aacgagatte tccgtettac ccecggetgea ggacaaacgt gcatggagta 840 cctaggtcct tacaccaaat ttgctggagg ccgaatcctce acceccgatg ctaccgacat 900 gtgcgaattc tgcgctgttg ctgacacgga caccttectg aagggggtta atatcatcett 960 tgatgaacgc tggagaaaca tcggcatttt gttcggatat attgecttca acatggtcgg 1020 teccateggc ttgtattgge ttetecgtet ccccaaaagg aaatctggeg tgaagcaggg 1080 acagcagcca € 1091
Claims (10)
1. Application of a trichodermin, characterized in that this trichodermin 1s used for preparation of drugs for treating skin diseases caused by Trichophyton mentagrophytes.
2. The application of claim 1, characterized in that, said trichodermin, as the metabolite of Trichoderma fungus, is preferentially from Trichoderma strain ZJUF0986.
3. The application of trichodermin of claim 1, characterized in that, the concentration of said trichodermin is greater than or equal to 10 pg/mL and smaller than 20 mg/mL.
4. The application of trichodermin of claim 1, characterized in that, said trichodermin inhibits colony growth of Trichophyton mentagrophytes.
5. The application of trichodermin of claim 4, characterized in that, the concentration of said trichodermin is greater than or equal to 10 pg/mL, and its inhibition rate for the colony growth of Trichophyton mentagrophytes is equal to or greater than 18%.
6. The application of trichodermin of claim 4, characterized in that, the concentration of said trichodermin is greater than or equal to 1000 ng/mL, and it totally inhibits the colony growth of Trichophyton mentagrophytes.
7. The application of trichodermin of claim 1, characterized in that, said trichodermin inhibits spore germination of Trichophyton mentagrophytes.
8. The application of trichodermin of claim 7, characterized in that, the concentration of said trichodermin is greater than or equal to 10 pg/mL, and its inhibition rate for the spore germination of Trichophyton mentagrophytes is equal to or greater than
69%.
a . . . nn , LU501586
9. The application of trichodermin of claim 7, characterized in that, the concentration of said trichodermin is greater than or equal to 100 ng/mL, and its inhibition rate for the spore germination of Trichophyton mentagrophytes is equal to or greater than
91%.
10. A method for regulating and controlling the function of trichodermin on Trichophyton mentagrophytes, characterized in that, this method comprises the following contents: By lowering the expression of gene CDRI1 or knocking out the gene CDR], the function of trichodermin for inhibiting Trichophyton mentagrophytes is enhanced.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU501586A LU501586B1 (en) | 2022-03-03 | 2022-03-03 | Application of a Trichodermin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| LU501586A LU501586B1 (en) | 2022-03-03 | 2022-03-03 | Application of a Trichodermin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| LU501586B1 true LU501586B1 (en) | 2023-09-11 |
Family
ID=88068169
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| LU501586A LU501586B1 (en) | 2022-03-03 | 2022-03-03 | Application of a Trichodermin |
Country Status (1)
| Country | Link |
|---|---|
| LU (1) | LU501586B1 (en) |
-
2022
- 2022-03-03 LU LU501586A patent/LU501586B1/en active IP Right Grant
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| FG | Patent granted |
Effective date: 20230911 |