Screening T-DNA Insertion Mutants With Hyper-osmosensitivity Phenotype And Study On Relationship Between Related Genes And Virulence In Aspergillus Fumigatus

Aspergillus fumigatus is ubiquitous, saprophytic fungi, widespread in naturalhabitat such as compost, decaying organic matter and soil. The conidia are very small(diameter of2~3μm).They can be spread through the air and have a longer survivaltime. It is estimated that an individual inhales several hundred conidia per day.Thesespores can be through the nose, trachea, bronchi end and reach the alveoli.In immunocompetent hosts, the non-specific and specific immune responses caneffectively remove these spores. However,with the recent clinical bone marrowtransplantation, organ transplantation and chemotherapy treatment carried out,leukemia,follicular fibrosis, increasing of patients infected with HIV, steroidhormones, corticosteroids and immunosuppressive agents cause impaired immunefunction, reduced defense capabilities, immunocompromised individuals cannot resistadhesion and invasion of conidia,resulting in infections caused by A. fumigatus withmortality rate has been above50%.1. Agrobacterium tumefaciens-mediated transformation system of AspergillusfumigatusThere is a tumor-inducing plamid (Ti plasmid) in A.tumefaciens. When plantcells are infected, T-DNA (T-DNA) located on Ti plasmid can be randomly integratedinto the chromosome, which lead to the overbalance of cell growth and formation ofcrown gall tumors. We use Agrobacterium tumefaciens-mediated T-DNA insertionalmutagenesis (ATMT) to build and obrain3205strains Aspergillus fumigatus insertionmutants (AFM). With mostly single locus insertion, use of HI-TAIL PCR and BLASTmethod, it can get a lot of single gene deletion mutants to study gene functionassociated.2.Screening T-DNA insertion mutants with hyper-osmosensitivity phenotypeTake advantage of inorganic ions NaCl constitute a high osmotic pressure,flatscreened the T-DNA insertion mutants at1.25M NaCl concentrations which is distinguish from the wild-type in growth,conidiation and hyphal morphology.Thereare320in2086mutants which have such changes and form a T-DNA insertionmutants sublibrary with sensitivity to hyperosmotic stress. HI-TAIL-PCR and BLASTidentified gene locus, a total of108mutants have been sequenced. The results showedthat these gene are related with osmotic sensor,membrane channel,energy metabolismand cell cycle.3. Construction of Aspergillus fumigatus metal homeostatis protein bsd2knockout mutant strain and complemented strainAFM3217has a phenotype of lower colony diameter,bleaching, reduced conidiaproduction at1.25M NaCl concentration.Based on HI-TAIL-PCR and BLAST,theinterrupted sequence is located upstream of bsd2determining gene,which may be theTATA box in promoter region. It prevents the binding of transcription factors to thepromoter region of the gene. Similar phenotypres were seen in bsd2knockout strainand AFM3217. Complemented strain is consistent with wild-type in phenotypes.4. Virulence and drug susceptibility testingAspergillus fumigatus infection in Caenorhabditis elegans model showedAFM3217was significantly decreased in virulence. Clinical laboratory used by theU.S. National Committee for Standardization (CLSI, former NCCLs) programenacted microdilution method (M38-A2), it shows that there is no difference betweenAFM3217and the wide type in sensitivity to fluconazole, itraconazole, voriconazole,posaconazole, amphotericin B, caspofungin, terbinafine, pimagedine.