Nahar Pallavi, Ghormade Vandana, Deshpande Mukund V
Biochemical Sciences Division, National Chemical Laboratory, Pashan Road, Pune 411 008, India.
J Invertebr Pathol. 2004 Feb;85(2):80-8. doi: 10.1016/j.jip.2003.11.006.
The possible contribution of extracellular constitutively produced chitin deacetylase by Metarhizium anisopliae in the process of insect pathogenesis has been evaluated. Chitin deacetylase converts chitin, a beta-1,4-linked N-acetylglucosamine polymer, into its deacetylated form chitosan, a glucosamine polymer. When grown in a yeast extract-peptone medium, M. anisopliae constitutively produced the enzymes protease, lipase, and two chitin-metabolizing enzymes, viz. chitin deacetylase (CDA) and chitosanase. Chitinase activity was induced in chitin-containing medium. Staining of 7.5% native polyacrylamide gels at pH 8.9 revealed CDA activity in three bands. SDS-PAGE showed that the apparent molecular masses of the three isoforms were 70, 37, and 26 kDa, respectively. Solubilized melanin (10microg) inhibited chitinase activity, whereas CDA was unaffected. Following germination of M. anisopliae conidia on isolated Helicoverpa armigera, cuticle revealed the presence of chitosan by staining with 3-methyl-2-benzothiazoline hydrazone. Blue patches of chitosan were observed on cuticle, indicating conversion of chitin to chitosan. Hydrolysis of chitin with constitutively produced enzymes of M. anisopliae suggested that CDA along with chitosanase contributed significantly to chitin hydrolysis. Thus, chitin deacetylase was important in initiating pathogenesis of M. anisopliae softening the insect cuticle to aid mycelial penetration. Evaluation of CDA and chitinase activities in other isolates of Metarhizium showed that those strains had low chitinase activity but high CDA activity. Chemical assays of M. anisopliae cell wall composition revealed the presence of chitosan. CDA may have a dual role in modifying the insect cuticular chitin for easy penetration as well as for altering its own cell walls for defense from insect chitinase.
已评估了绿僵菌在昆虫致病过程中细胞外组成型产生的几丁质脱乙酰酶的可能作用。几丁质脱乙酰酶将几丁质（一种β-1,4-连接的N-乙酰葡糖胺聚合物）转化为其脱乙酰形式的壳聚糖（一种葡糖胺聚合物）。当在酵母提取物-蛋白胨培养基中生长时，绿僵菌组成型产生蛋白酶、脂肪酶和两种几丁质代谢酶，即几丁质脱乙酰酶（CDA）和壳聚糖酶。在含几丁质的培养基中诱导产生几丁质酶活性。在pH 8.9条件下对7.5%天然聚丙烯酰胺凝胶进行染色，结果显示三条带具有CDA活性。SDS-PAGE表明三种同工型的表观分子量分别为70、37和26 kDa。溶解的黑色素（10微克）抑制几丁质酶活性，而CDA不受影响。绿僵菌分生孢子在离体棉铃虫上萌发后，通过用3-甲基-2-苯并噻唑啉腙染色，角质层显示出壳聚糖的存在。在角质层上观察到壳聚糖的蓝色斑块，表明几丁质已转化为壳聚糖。用绿僵菌组成型产生的酶对几丁质进行水解表明，CDA与壳聚糖酶一起对几丁质水解有显著贡献。因此，几丁质脱乙酰酶在绿僵菌引发致病过程中很重要，它可软化昆虫角质层以帮助菌丝体穿透。对其他绿僵菌分离株中CDA和几丁质酶活性的评估表明，那些菌株的几丁质酶活性低但CDA活性高。对绿僵菌细胞壁成分的化学分析显示存在壳聚糖。CDA可能具有双重作用，一是修饰昆虫表皮几丁质以便于穿透，二是改变自身细胞壁以抵御昆虫几丁质酶。
