| Endophytic fungi are treasure trove of natural products that produce identical or similar pharmacologically active compounds during synergistic evolution with their host plants.Endophytic fungi are easy to cultivate and biologically active,with chemically diverse metabolites,broadening the source of novel antibacterial substances for screening,endophytic fungi provides a novel strategy for the treatment of infectious diseases caused by bacteria.Ageratina adenophora is at the top of the list of China’s first invasive alien species published by the National Environmental Protection Administration and the Chinese Academy of Sciences,but it is used as a traditional medicine in Nigeria,Sikkim,India and some area in China.Previous study in our laboratory confirmed that the extract from Ageratina adenophora has good antibacterial effect on pathogenic bacteria such as Escherichia coli,Staphylococcus aureus and gypsum-like microsporidia.Therefore,this study was conducted to isolate and identify culturable endophytic fungi from Ageratina adenophora in Luoji Mountain Nature Reserve.Firstly,to screen out strains with significant antibacterial activity.Secondly to select the endophytic fungi with significant changes in metabolic composition before and after the addition of epi-modifiers,and to isolate purify and structurally identify the antibacterial active components in ethyl acetate site.Finally,the more active compounds were selected for the study of the molecular mechanism of antibacterial action,to provide a reference basis for the exploration of their action targets.The main research contents and results are as follows::1.Isolation and identification of endophytic fnngi and screening of antibacterial activity in Ageratina adenophoraThe endophytic fungi of Ageratina adenophora from the Luoji Mountain Nature Reserve were isolated and identified by surface disinfection procedure,tissue block isolation method,lactophenal cotton blue stain microscopy and ITS sequencing.Strains with antibacterial activity(Escherichia coli,Salmonella,Streptococcus lactis and Staphylococcus aureus)were screened by agar diffusion method and micro broth dilution method,and chemical composition of their metabolites were analyzed by liquid chromatography-mass spectrometry(LC-MS).RESULTS:(1)According to the preliminary identification of fungal morphology,combined into 124 strains(including 56 isolated from leaves,36 isolated from roots and 32 isolated from stems),belonging to 3 phyla,7 classes,12 orders,16 families and 26 genera.(2)25 endophytic fungi had antibacterial activity against more than one pathogenic fungus,whereas 5 endophytic fungal extracts had antibacterial activity against at least 1 clinically isolated multi-drug resistant bacteria.(3)DCL06,DCL09 and DCL44 strains showed strong antibacterial activity against MRSA,and their activities may be related to organic acids and flavonoids.The above results indicate that Ageratina adenophora contains a rich variety of endophytic fungi with antibacterial activity,hence,can be used as a microbial source of novel antibacterial substances.2.Identification of secondary metabolites of endophytic fungns DCL44 and their antibacterial activityDCL06,DCL09 and DCL44 were induced by vorinostat and other five epigenetic modifiers,respectively,and the significant changes in the strains’ chemical composition before and after epigenetic modification were analyzed using HPLC.The endophytic fungal metabolites were separated by lysis,silica gel column chromatography and recrystallization,and their antibacterial activities were determined agar diffusion and micro-broth dilution methods.RESULTS:The isolated monomeric compounds of DCL44 metabolites were cyclic peptides or amide alkaloids,namely Cyclo(L-Len-trans-4hydroxy-L-Pro,Cyclo(L-Phe-trans-4-hydroxy-L-Pro),Suberanilic acid,N1Phenylsuberamide and 4-(5-oxotetrahydrofuran-2-yl)-N-phenylbutanamide.Among the five compounds,the MIC value of Suberanilic acid against MRS A ATCC43300 was 32μg/mL.However,the other 4 compounds had poor antibacterial activity(MIC value ≥256μg/mL)against the tested pathogens(E.coli,Salmonella and MRS A).3.In vitro antibacterial activity of Suberanilic acid against MRSAThe in vitro antibacterial properties of Suberanilic acid against MRSA were investigated by the minimum inhibitory concentration,growth curve and combined drug sensitivity tests and extracellular alkaline phosphatase content,LIVE/DEAD assay,electron microscopic ultrastructural observation and SDS-PAGE technique.RESULTS:(1)The MIC and MBC values of Suberanilic acid against MRS A ATCC43300 and clinical isolates were 16-128 μg/mL and 32-256 μg/mL,respectively,and the MIC and 2 MIC had sustained bactericidal effects.(2)Suberanilic acid treatment on MRSA result in a significant increase in supernatant alkaline phosphatase activity,nucleic acid and protein leakage(P<0.05).In addition,the results also showed a significant differences(P<0.01)between the green fluorescence optical density and the red fluorescence optical density for MRSA ATCC43300 bacteriophage in the Suberanilic acid treatment groups of 1/2 MIC and 2 MIC,however,there was no significant difference(P>0.05)between green fluorescence optical density and red fluorescence optical density in the Suberanilic acid treatment group of MIC.Moreover,the ultrastructure of the bacterium showed that the MRSA cell wall was separated,the cell membrane was ruptured,and the cytoplasmic contents were lost and formed empty vesicle structure.(3)The grayscale values of approximately 15 kDa size protein bands in the 1/2×MIC and 1×MIC Suberanilic acid treated groups showed highly significant differances compared with the control group(P<0.01).The above results indicate that Suberanilic acid has strong antibacterial activity against MRSA at a concentration-dependent manner via inhibition of MRSA protein expression.4.Proteomic analysis of MRSA under the action of Suberanilic acid based on TMT technologyTMT(Tandem Mass Tag)-LC-MS/MS(Liquid chromatography-mass spectrometry/mass spectrometry)technology were used to analyze the sub-inhibitory concentration(1/2 MIC)of both the Suberanilic acid-treated and the control groups for significant changes in differentially expressed proteins(388 up-regulated and 386 down-regulated),combined with bioinformatics methods to analyze their functions,and to validate the proteomic results by parallel reaction monitoring.RESULTS:(1)The differentially expressed proteins were annotated by the Gene Ontology Database,and the differentially expressed proteins were mainly involved in 10 biological processes such as organic acid metabolism and carboxylic acid metabolism;10 cellular components such as ribosomes,cell walls and cytoplasm;and 6 molecular functions such as catalytic activity and binding action.(2)The KEGG pathway database annotation enrichment analysis showed that the differentially expressed proteins were mainly enriched in the ABC transporter system,Pyrnvic acid metabolism,ribosome,and tricarboxylic acid cycle pathways.Conclusion:Among the 124 morphotypes of endophytic fungi isolated from Ageratina adenophora,DCL44 had the strongest antibacterial activity against MRSA,and the main antibacterial active component was Suberanilic acid,and its mechanism may be through affecting the synthesis of ribosomessuch as rpsQ and rpmD,inhibition of expression of key proteins such as cycB and pstS in the ABC transport system and inhibition of metabolism of amino acids such as purA,argG and glmS,which in turn inhibits MRS A energy activity and thus MRS A growth. |
