Use of Bulk Segregant Analysis for Determining the Genetic Basis of Azole Resistance in the Opportunistic Pathogen Aspergillus fumigatus.
Use of Bulk Segregant Analysis for Determining the Genetic Basis of Azole Resistance in the Opportunistic Pathogen Aspergillus fumigatus.
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DOI：
10.3389/fcimb.2022.841138
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发表时间：
2022
影响因子：
5.7
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中科院分区：
文献类型：
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2009年描述了机会性真菌病原体烟曲霉的有性周期，首次打开了利用有性杂交技术进行遗传分析的可能性。本研究旨在评估“整体分离分析”(BSA)技术是否可以与下一代测序相结合来研究烟曲霉单基因性状的潜在基础。选择对唑类抗真菌药物伊曲康唑的抗性作为模型，开发了一条专门的生物信息学管道，以识别与敏感子代库和亲本相比，抗性子代库和抗性亲本之间存在差异的SNPs。将一株对伊曲康唑耐药的临床分离株与牛血清白蛋白敏感亲本及F1代杂交。此外，还评估了利用回交和增加子代池中的数量作为提高BSA效率的方法。使用40个子代的F1基因库，当与A1163参考基因组比对时，识别出123个候选基因，SNPs分布在几个重叠群中。连续几轮回交增强了识别特定基因和基因组区域的能力，第三轮回交的后代(每个池使用40个)的BSA识别了46个SNPs基因，第六轮回交的后代的BSA识别了20个基因的SNPs，这些基因位于基因组的一个292 kb区域。每个池增加80个后代的使用也提高了BSA的分辨率，29个基因显示了利用第二次回交的后代检测到的不同敏感和抗性群体之间的SNPs，大多数变异位于相同的292kb区域。对292kb区域的进一步生物信息学分析证实了cyp51a基因变异的存在，导致了CYP51a蛋白的蛋氨酸到赖氨酸(M220K)的变化，这被认为是观察到的伊曲康唑耐药性的原因。讨论了牛血清白蛋白在烟曲霉菌遗传分析中的应用前景。
A sexual cycle was described in 2009 for the opportunistic fungal pathogen Aspergillus fumigatus, opening up for the first time the possibility of using techniques reliant on sexual crossing for genetic analysis. The present study was undertaken to evaluate whether the technique ‘bulk segregant analysis’ (BSA), which involves detection of differences between pools of progeny varying in a particular trait, could be applied in conjunction with next-generation sequencing to investigate the underlying basis of monogenic traits in A. fumigatus. Resistance to the azole antifungal itraconazole was chosen as a model, with a dedicated bioinformatic pipeline developed to allow identification of SNPs that differed between the resistant progeny pool and resistant parent compared to the sensitive progeny pool and parent. A clinical isolate exhibiting monogenic resistance to itraconazole of unknown basis was crossed to a sensitive parent and F1 progeny used in BSA. In addition, the use of backcrossing and increasing the number in progeny pools was evaluated as ways to enhance the efficiency of BSA. Use of F1 pools of 40 progeny led to the identification of 123 candidate genes with SNPs distributed over several contigs when aligned to an A1163 reference genome. Successive rounds of backcrossing enhanced the ability to identify specific genes and a genomic region, with BSA of progeny (using 40 per pool) from a third backcross identifying 46 genes with SNPs, and BSA of progeny from a sixth backcross identifying 20 genes with SNPs in a single 292 kb region of the genome. The use of an increased number of 80 progeny per pool also increased the resolution of BSA, with 29 genes demonstrating SNPs between the different sensitive and resistant groupings detected using progeny from just the second backcross with the majority of variants located on the same 292 kb region. Further bioinformatic analysis of the 292 kb region identified the presence of a cyp51A gene variant resulting in a methionine to lysine (M220K) change in the CYP51A protein, which was concluded to be the causal basis of the observed resistance to itraconazole. The future use of BSA in genetic analysis of A. fumigatus is discussed.
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影响因子：
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通讯作者：
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影响因子：
3.7
作者：
Baird NA;Etter PD;Atwood TS;Currey MC;Shiver AL;Lewis ZA;Selker EU;Cresko WA;Johnson EA
通讯作者：
Johnson EA
影响因子：
11.8
作者：
Denning, David W.;Park, Steven;Perlin, David S.
通讯作者：
Perlin, David S.
影响因子：
1.2
作者：
Cingolani, Pablo;Platts, Adrian;Ruden, Douglas M.
通讯作者：
Ruden, Douglas M.
影响因子：
11.8
作者：
Hare, Rasmus K.;Gertsen, Jan B.;Arendrup, Maiken C.
通讯作者：
Arendrup, Maiken C.