Rajendran Ranjith, Williams Craig, Lappin David F, Millington Owain, Martins Margarida, Ramage Gordon
School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom.
Eukaryot Cell. 2013 Mar;12(3):420-9. doi: 10.1128/EC.00287-12. Epub 2013 Jan 11.
Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.
烟曲霉已被证明能形成与适应性抗真菌耐药机制相关的生物膜。这些机制包括多药外排泵、热休克蛋白和细胞外基质（ECM）。ECM是微生物生物膜的关键结构和保护成分，在细菌中已被证明含有细胞外DNA（eDNA）。因此，我们推测烟曲霉生物膜也具有作为ECM一部分的eDNA，并发挥功能性作用。荧光显微镜和定量PCR分析表明存在eDNA，其释放具有阶段依赖性（8小时<12小时<24小时<48小时）。随机扩增多态性DNA（RAPD）PCR显示eDNA与基因组DNA相同。用DNase处理会破坏生物膜的结构完整性。生化和转录分析表明，随着生物膜成熟，几丁质酶活性以及几丁质酶（自溶标志物）的mRNA水平显著上调，并且抑制几丁质酶会影响生物膜的生长和稳定性，从机制上表明可能涉及自溶。最后，使用棋盘法分析表明，用DNase联合两性霉素B和卡泊芬净处理生物膜可显著提高抗真菌药敏性。总的来说，这些数据表明eDNA是烟曲霉ECM的重要结构成分，通过自溶释放，这对于抵御包括抗真菌治疗在内的环境压力很重要。
