dlt操纵子赋予苏云金芽胞杆菌对阳离子抗菌肽的抗性和对昆虫的毒力

The dlt operon in Bacillus thuringiensis confers resistance to cationic antimicrobial peptides and virulence to insect

摘要:

[目的]革兰氏阳性菌中的dlt操纵子编码细胞壁中磷壁酸发生D-丙氨酰化修饰所必需的酶。D-丙氨酰化使细胞表面产生正电荷，并因此排斥带正电的分子，例如阳离子抗菌肽，从而赋予对宿主的抗性。本文中，我们研究了dlt操纵子对苏云金芽胞杆菌表型性状的影响及在对昆虫毒力中发挥的作用。[方法]通过同源重组构建了ΔdltA_Bt基因缺失突变株，并对其进行形态学观察、表面电荷差异分析、抗逆性分析和毒力测定。[结果]结果表明，dltA的失活显著降低了细胞表面的净负电荷，对阳离子抗菌肽（多粘菌素B和溶菌酶）的抗性和碱耐受性显著下降。同时，ΔdltA_Bt的生长曲线发生明显改变，细胞表面粗糙且形状不规则，生物膜形成减少和群游运动能力增强。此外，dltA的失活降低了对昆虫中肠上皮细胞的粘附能力，并减弱了对家蚕的毒力。[结论]研究结果表明，dlt操纵子介导的磷壁酸发生D-丙氨酰化修饰与苏云金芽胞杆菌的许多表型性状密切相关，并且在苏云金芽胞杆菌对昆虫的致病性及抵抗昆虫体液免疫保护中具有重要作用。

Abstract:

[Objective] The dlt operon in Gram-positive bacteria encodes enzymes that are necessary for the modification of D-alanylation of teichoic acids in cell wall. D-alanylation generates net positive charge on cell surface and, as a consequence, repulses the positively charged molecules, such as cationic antimicrobial peptides, thereby confers resistance to host animal. Here, we investigated the impact of dlt operon on phenotypic traits of Bacillus thuringiensis and role in virulence to insect. [Methods] We constructed the loss-of-function mutant of dlt by homologous recombination technique, and performed its morphological observation, surface charge difference analysis, stress resistance analysis and cell experiment.[Results] The results revealed that inactivation of dltA significantly decreased net negative charge of cell wall, drastically impaired the resistance of Bacillus thuringiensis to cationic antimicrobial peptides (polymyxin B and lysozyme) and alkaline. ΔdltA_Bt mutant displayed an obviously altered profile of growth curve, irregular shape and rough surface of cell, decreased biofilm formation and increased swarming motility. Moreover, inactivation of dltA significantly decreased adhesion ability to mid-gut epithelial cell of insect, and greatly attenuated virulence to Bombyx mori. [Conclusion] These findings provide evidence that D-alanylation of TAs mediated by dlt operon is closely correlated to many phenotypic traits of Bt, and has putative roles in the pathogenicity of B. thuringiensis to insect and the protection of B. thuringiensis from insect humoral immunity.