Study On Virulence Factor And Host Immune Response Mechanism Of Aspergillus Lentulus Isolated From COPD Patient

Objective:To investigate the morphology,molecular biology and in vitro drug sensitivity of Aspergillus lentulus(A.lentulus),the first strain isolated from COPD patients in China,and further study its virulence and the expression of virulence factors in animal models after infection.To explore its possible host immune response mechanism by testing the expression of Caspase-1 and terminal inflammatory factors.Methods:The morphological characteristics of the isolate from the sputum of a patient with COPD was observed under optical microscope.Theβ-tubulin(560bp)gene was amplified and blasted to complete gene identification.In vitro drug sensitivity test was performed according to CLSI M38-A2 standard.Using Aspergillus fumigatus and Candida albicans as control,the A.lentulus bacterial solution at concentration of 1×10~6CFU was selected to inject into the wax moth larvae,and the survival rate,melanization score and activity score of larvae were observed and compared at different time points.The expression of(1,3)-β-D glucan and galactomannan in larvae tissue were detected by colorimetry and enzymatic method.Mouse dendritic cells were treated with A.lentulus,Aspergillus fumigatus and Candida albicans at different MOI values:0,1,5,10,20,and IL-βin the supernatant was detected by ELISA.Finally,1×10~6CFU of A.lentulus was injected into the larvaes,and the m RNA expression level of Caspase-1 and TNF-αin the larvaes after infection was detected by q RT-PCR.Results:1)Observation of the isolated strains under microscope:A.lentulus were growing in the form of white,flocculent colonies and could not grow at all at 48℃.Hyaline branched hypha can be seen with few spores;2)Gene identification results:the colony showed 99%homology with standard A.lentulus strain;3)The results of in vitro antifungal susceptibility testing:amphotericin B(MIC 2μg/ml),5-fluorocytosine(MIC64μg/ml),fluconazol(MIC>64μg/ml),itraconazole(MIC 0.5μg/ml),voriconazole(MIC1μg/ml),miconazole(MIC 4μg/ml),micafungin(MIC≤0.015μg/ml).Except for miconazole(MIC 2μg/ml)and micafungin(MIC≤0.03μg/ml),the antimicrobial susceptibility results of the standard strain FH5T are the same as those of the patient strain;4)In the infection model of wax moth larvae,the larvae infected with Aspergillus fumigatus and Candida albicans all died within 24-48 hours,while the melanization score,survival rate score and activity score of the larvae infected with A.lentulus only decreased by 57.5%,73.3%,and 53.3%respectively compared with the control group at 48 hours.There is no significant difference of(1,3)β-D-glucan level in the A.lentulus patient strain group and the standard strain group compared with the PBS control group(P>0.05).The galactomannan level of A.lentulus patient strain group and standard strain group was increased significantly compared with the PBS control group(P<0.05),while no significant difference was showed between A.lentulus patient strain group and standard strain group(P>0.05);5)The candida albicans,Aspergillus fumigatus and A.lentulus can induce IL-1βexpression in mouse dendritic cells.When cells were treated with Candida albicans with MOI=20 for 48 hours,the IL-1βlevel was higher than other time points and concentrations.When treated with Aspergillus fumigatus for 12 hours with MOI=10,IL-1βsignificantly increased compared with MOI=0(P<0.01).Compared with MOI=0,there was a significant induction of IL-1βwith A.lentulus treatment for 48 hours at MOI=1.Starting from 12 hours,the IL-1βlevel induced by Aspergillus fumigatus was significantly higher than that of A.lentulus patient strains at same time and concentration(P<0.01).Both of A.lentulus patient strain and standard strain can induce m RNA of Caspase-1 and TNF-αexpression in larvae infection model(P<0.05),and no significant difference was observed between the two infection groups(P>0.05).Conclusion:1)The A.lentulus isolated from COPD patient in China is mainly white,flocculent with hyaline branched hypha and few spores,which cannot grow at all at 48℃.The strain was sensitive to micafungin,while showed high MIC to amphotericin B,5-flucytosine,fluconazole and miconazolein in vitro;2)The A.lentulus isolated from patient was less virulent than Aspergillus fumigatus;3)A.lentulus infection can cause the release of IL-βin mouse dendritic cells,but its less virulent than Candida albicans and Aspergillus fumigatus under the same conditions;4)Galactomannan can be used as a monitoring marker for early invasive infection of A.lentulus;5)The NLRP3/Caspase-1 inflammasome pathway is involved in the immune response of A.lentulus infected host,which is accompanied by an increase of TNF-α.