Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.
Membrane-Bound PenA β-Lactamase of Burkholderia pseudomallei.
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类鼻疽伯克霍尔德氏菌的膜结合 PenA β-内酰胺酶。
DOI：
10.1128/aac.02444-15
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发表时间：
2015
影响因子：
4.9
通讯作者：
Schweizer,HerbertP
中科院分区：
文献类型：
--
作者：
Randall,LinnellB;Dobos,Karen;Papp-Wallace,KrisztinaM;Bonomo,RobertA;Schweizer,HerbertP
类鼻疽伯克霍尔德氏菌是类鼻疽的病原体，临床表现多样，是一种难治性疾病。头孢他啶等β-内酰胺类抗生素对类鼻疽病治疗的成功至关重要。对头孢他啶耐药的临床分离株已有报道，最常见的机制是点突变影响染色体编码的A类内酰胺酶β-内酰胺酶的表达或关键氨基酸残基。我们以前发现Pena是通过双精氨酸转位酶系统输出的，并与球体部分相关。我们现在证明Pena是一种膜结合的脂蛋白。在膜组分中发现蛋白质和伴随的β-内酰胺酶活性，并可用Triton X-114提取。用球霉素处理表达PenA的假鼻疽杆菌细胞，会导致前脂蛋白的积累。对提取的膜蛋白进行了质谱分析，发现了一个蛋白质峰，其质量与三酰化的Pena蛋白一致。23位脂盒半胱氨酸突变为丝氨酸残基，导致β-内酰胺酶活性丧失，检测到的PenAC23S蛋白缺失。伴随的异亮氨酸到丙氨酸的变化伴随在PenAC23S信号肽加工位点的第20位，导致非脂蛋白(PenAI20A C23S)被信号肽I加工，并显示β-内酰胺酶活性。表达该蛋白的假鼻疽杆菌菌株的抗性图谱与表达野生型Pena的同基因菌株的图谱难以区分。数据表明，PENA膜缔合不是抗性所必需的，必须用于另一目的。
Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23Sprotein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23Smutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose.