Metalloprotease production by Paenibacillus larvae during the infection of honeybee larvae
Metalloprotease production by Paenibacillus larvae during the infection of honeybee larvae
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DOI：
10.1099/mic.0.044321-0
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发表时间：
2011-05-01
期刊：
影响因子：
2.8
通讯作者：
Zunino, Pablo
中科院分区：
文献类型：
--
作者：
Antunez, Karina;Arredondo, Daniela;Zunino, Pablo
美洲蜜蜂幼虫病是一种世界性分布的细菌性疾病，影响意大利蜜蜂的幼虫。病原体是革兰氏阳性孢子形成细菌幼虫类芽孢杆菌。一些作者提出，幼虫疟原虫分泌金属蛋白酶，参与感染后发生的幼虫降解。本工作的目的是评估在幼虫感染期间由P. larvae产生的金属蛋白酶。首先，在幼虫拟青霉基因组中鉴定编码金属蛋白酶的完整基因，并通过PCR在来自不同地理区域的幼虫拟青霉分离物的集合中评估其分布。扩增、克隆、表达重组金属蛋白酶基因，纯化重组金属蛋白酶基因，制备抗金属蛋白酶抗体，免疫荧光和荧光原位杂交检测金属蛋白酶的表达。编码幼虫疟原虫金属蛋白酶的基因广泛分布在来自乌拉圭和阿根廷不同地理来源的分离株中。金属蛋白酶存在于幼虫营养细胞内、孢子表面并分泌到外部培养基中。该蛋白在感染蜜蜂幼虫的体内也得到了证实，它能够水解乳蛋白，这表明它可能参与了幼虫的降解。这项工作有助于了解具有重要经济意义的细菌的致病机制，并且是表征潜在的P.幼虫毒力因子的一步。
American foulbrood is a bacterial disease of worldwide distribution that affects larvae of the honeybee Apis mellifera. The causative agent is the Gram-positive, spore-forming bacterium Paenibacillus larvae. Several authors have proposed that P. larvae secretes metalloproteases that are involved in the larval degradation that occurs after infection. The aim of the present work was to evaluate the production of a metalloprotease by P. larvae during larval infection. First, the complete gene encoding a metalloprotease was identified in the P. larvae genome and its distribution was evaluated by PCR in a collection of P. larvae isolates from different geographical regions. Then, the complete gene was amplified, cloned and overexpressed, and the recombinant metalloprotease was purified and used to generate anti-metalloprotease antibodies.Metalloprotease production was evaluated by immunofluorescence and fluorescence in situ hybridization. The gene encoding a P. larvae metalloprotease was widely distributed in isolates from different geographical origins in Uruguay and Argentina. Metalloprotease was detected inside P. larvae vegetative cells, on the surface of P. larvae spores and secreted to the external growth medium. Its production was also confirmed in vivo, during the infection of honeybee larvae.This protein was able to hydrolyse milk proteins as described for P. larvae, suggesting that could be involved in larval degradation. This work contributes to the knowledge of the pathogenicity mechanisms of a bacterium of great economic significance and is one step in the characterization of potential P. larvae virulence factors.