Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae
Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae
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DOI：
10.1016/j.gene.2003.09.031
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发表时间：
2004-01-07
期刊：
影响因子：
3.5
通讯作者：
St Leger, RJ
中科院分区：
文献类型：
--
作者：
Bagga, S;Hu, G;St Leger, RJ
真菌分泌枯草杆菌蛋白酶以获取营养并突破宿主屏障。在此，我们试图对昆虫病原绿僵菌中枯草杆菌蛋白酶的多样性进行全面表征。表达序列标签（EST）分析表明，绿僵菌小孢变种（菌株2575）的一个广谱宿主菌株在昆虫表皮上生长时表达11种枯草杆菌蛋白酶，这是在所有真菌中报道的枯草杆菌蛋白酶数量最多的。聚合酶链反应从具有多个宿主的第二个菌株（菌株820）中扩增出其中10种直系同源物，从蝗虫专化型绿僵菌蝗变种（菌株324）中扩增出7种。基于序列相似性和外显子 - 内含子结构的分析将绿僵菌枯草杆菌蛋白酶分为四个簇——一类I（“细菌型”）枯草杆菌蛋白酶（Pr1C），以及三个蛋白酶K样的二类枯草杆菌蛋白酶簇：胞外亚家族1（Pr1A、Pr1B、Pr1G、Pr1I和Pr1K）、胞外亚家族2（Pr1D、Pr1E、Pr1F和Pr1J）以及一种胞内枯草杆菌蛋白酶（Pr1H）。对来自其他属的同源序列的系统发育分析表明，这种将蛋白酶K样枯草杆菌蛋白酶细分为三个亚家族的情况发生在主要真菌谱系分化之前。然而，在绿僵菌枯草杆菌蛋白酶的进化过程中，多样化一直在持续，有证据表明在绿僵菌小孢变种和绿僵菌蝗变种分化之后发生了基因复制事件。比较一个谱系内以及不同谱系间Pr1同工酶的比对和非同义/同义替换率表明，虽然枯草杆菌蛋白酶的总体分化符合中性预期，但参与催化的氨基酸受到强烈的选择约束。这表明每个Pr1旁系同源物都对病原体的适应性有贡献。此外，同源建模预测了Pr1在其次级底物特异性、对表皮的吸附特性以及碱性稳定性方面的差异，这表明了功能上的差异。（C）2003 Elsevier B.V.保留所有权利。
Fungi secrete subtilisin proteinases to acquire nutrients and breach host barriers. Here we sought a global characterization of the diversity of subtilisins in the insect pathogen Metarhizium anisopliae. Expressed sequence tag (EST) analyses showed that a broad host range strain of M. anisopliae sf. anisopliae (strain 2575) expressed 11 subtilisins during growth on insect cuticle, the largest number of subtilisins reported from any fungus. Polymerase chain reaction amplified 10 of their orthologs from a second strain with multiple hosts (strain 820) and seven from the locust specialist M anisopliae sf. acridum (strain 324). Analyses based on sequence similarities and exon-intron structure grouped M anisopliae subtilisins into four clusters-a class I ("bacterial") subtilisin (Pr1C), and three clusters of proteinase K-like class II subtilisins: extracellular subfamily 1 (Pr1A, Pr1B, Pr1G, Pr1I and Pr1K), extracellular subfamily 2 (Pr1D, Pr1E, Pr1F and Pr1J) and an endocellular subtilisin (Pr1H). Phylogenetic analysis of homologous sequences from other genera revealed that this subdivision of proteinase K-like subtilisins into three subfamilies preceded speciation of major fungal lineages. However, diversification has continued during the evolution of Metarhizium subtilisins with evidence of gene duplication events after divergence of M. anisopliae sf. anisopliae and M anisopliae sf. acridum. Comparing alignments and nonsynonymous/synonymous rates for Pr1 isoenzymes within a lineage and between lineages showed that while overall divergence of subtilisins followed neutral expectations, amino acids involved in catalysis were under strong selective constraint. This suggests that each Pr1 paralog contributes to the pathogens fitness. Furthermore, homology modeling predicted differences between the Pr1's in their secondary substrate specificities, adsorption properties to cuticle and alkaline stability, indicative of functional differences. (C) 2003 Elsevier B.V. All rights reserved.