Analysis Of The Active Components Of A Novel Species Of Actinomycete Inhibiting Candida Albicans Biofilm Formation And Preliminary Study On Its Mechanism

Candida albicans is one of the major pathogen of nosocomial fungal infection.In recent years,with the wide application of broad-spectrum antibiotics,corticosteroids and immunosuppressants,the morbidity and mortality of fungal infections have been increasing year by year.The formation of biofilm?BF?helps the fungus evade immune system clearance and drug attack,making it more difficult to treat C.albicans infection.Therefore,the development of new antibacterial agents is urgent.Actinomycetes can produce abundant secondary metabolites,which is a treasure trove for mining biological active substances.In this experiment,rich microbial resources in southern Xinjiang were used to screen the strains that inhibit the formation of C.albicans BF,determine the taxonomic status,find activity-related genes through the analysis of genome sequencing information,clarify the composition of active secondary metabolites,and explore the anti-biofilm mechanism,to provide theoretical basis for the development of new anti-biofilm agents.1.Taking C.albicans as the target bacteria,170 strains of actinomycetes from flowing sand and continuous cultivated cotton fields were screened by microplate quantification assay method.The results showed that 6 strains of actinomycetes,including TRM43335,TRM43290 and TRM43259,could inhibit the formation of C.albicans BF.2.The strain TRM43335^T was polyphasically classified based on the apparent,chemical and genotypic characteristics,and identified as a new species of Streptomyces,and named as Streptomyces taklimakanensis.The strain formed abundant aerial mycelium,occasionally twisted and differentiated into spiral spore chains.Each spore of TRM43335^T was oval-shaped with a smooth surface and growing optimally at 37°C,pH 8 and in the presence of 1%?w/v?NaCl.DNA G+C content of the type strain was72.8 mol%.The major sugars were ribose,xylose,glucose,mannose,and galactose,and the principal phospholipids were diphosphatidylglycerol?DPG?,phosphatidylinositolmannoside?PIM?,phosphatidylglycerol?PG?,phosphatidylinositol?PI?,phosphatidylethanolamine?PE?,LL-diaminopimelic acid as diagnostic amino acids.The predominant menaquinone was MK-9?H₆?.The major cellular fatty acids were iso-C_16:16:1 H,anteiso-C_15:0,iso-C_16:0,anteiso-C_17:0,anteiso-C_17:17:1 w9c and iso-C_15:0.Analysis of 16S rRNA sequence showed that strain TRM43335^T had highest sequence similarity to Streptomyces desertarenae SYSU D8023^T 98.69%.The average nucleotide identity?ANI?relatedness between strain TRM43335^T and phylogenetically related strains Streptomyces desertarenae SYSU D8023^T were 89.23%,DNA-DNA hybridization?dDDH?value were 36.70%.The type strain is TRM43335^T?CCTCC AA2018052^T=KCTC 49254^T?.3.The complete genome of TRM43335^T strain was sequenced by Nanopore sequencing technology,and was reasonably classified and annotated by various bioinformatics analysis tools.Finally,the whole genome sequence with length of 6136714 bp was obtained,of which 5,163 genes were given explicit functional annotation.4.The protein was extracted from fermentation broth of strain TRM43335^T by ammonium sulfate precipitation method.Sephadex G-100 gel filtration chromatography and DEAE ion exchange chromatography were used for separation and purification,and sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?was used for detection.The activity detection results showed that fractions C,D,and E had good activity,and the subsequent physical and chemical factors were investigated.The results showed that the fractions were insensitive to heat and relatively stable to acids and bases,and the anti-biofilm activity decreased at PH 7.Metal ions and organic reagents had no promoting effect on biofilm inhibition.The inhibition mechanism of fraction proteins on formation of C.albicans BF was determined by milk plate experiment,surface hydrophobicity experiment and extracellular DNA degradation experiment.The results showed that fraction proteins C and D could hydrolyze extracellular proteins and reduce surface hydrophobicity.Protein E could hydrolyze extracellular proteins,reduce surface hydrophobicity,and also degrade extracellular DNA.