In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA).
In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA).
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DOI：
10.1371/journal.ppat.1006667
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发表时间：
2017-10
期刊：
影响因子：
6.7
通讯作者：
Clarke AJ
中科院分区：
文献类型：
--
作者：
Sychantha D;Jones CS;Little DJ;Moynihan PJ;Robinson H;Galley NF;Roper DI;Dowson CG;Howell PL;Clarke AJ
必需的细胞壁聚合物肽聚糖的O-乙酰化发生在大多数革兰氏阳性细菌病原体中，包括葡萄球菌属、链球菌属和肠球菌属。肽聚糖的这种修饰保护这些病原体免受先天免疫系统的溶菌酶的裂解作用，因此被认为是毒力因子。肽聚糖O-乙酰基转移酶A（OatA）是一种膜结合蛋白，其底物是不溶性肽聚糖细胞壁聚合物，因此对生物化学研究具有特殊的挑战性。OatA被预测为是双模块的，由连接到C-末端胞质外结构域的N-末端整合膜结构域组成。我们在此提出的第一个生化和动力学表征的C-末端催化结构域的OatA从两个重要的人类病原体，金黄色葡萄球菌和肺炎链球菌。使用假底物和新型生物合成制备的肽聚糖聚合物，我们表征了这两种酶不同的底物特异性。此外，C-末端结构域的高分辨率晶体结构揭示了SGNH/GDSL样水解酶折叠，其具有催化三联体的氨基酸，但具有非典型的含氧阴离子空穴结构。位点特异性置换证实了催化和含氧阴离子空穴残基的身份。提出了一个模型的O-乙酰化的肽聚糖，其中转运的乙酰基从细胞质来源的跨细胞质膜被催化的N-末端结构域的OatA为他们的转移到肽聚糖的C-末端结构域。OatA的结构-功能关系的研究提供了对这种细菌耐药机制的分子和机制的理解，为新的化疗探索开辟了前景，以增强对革兰氏阳性病原体的先天免疫保护。耐甲氧西林金黄色葡萄球菌（MRSA）、耐万古霉素肠球菌（VRE）和耐药肺炎链球菌（DRSP）等重要人类病原体的多重耐药性继续挑战临床医生并威胁感染患者的生命。在解决这一严重问题的几种方法中，开发拮抗剂使细菌感染对我们先天免疫系统的防御酶和蛋白质更敏感。一种这样的靶标是酶O-乙酰转移酶A（OatA）。这种胞外酶修饰细菌细胞壁的基本成分肽聚糖，从而使其对溶菌酶的溶解作用具有抗性，溶菌酶是我们抵御入侵病原体的第一道防线。在这项研究中，我们提出了第一个生化和结构表征的燕麦。使用S。金黄色葡萄球菌和作为模型系统，我们证明了OatA具有独特的底物特异性。我们还表明，OatA的催化结构域是一个结构同源的水解酶的超家族研究。它使用Ser-His-Asp催化三联体将乙酰基特异性转移到肽聚糖内胞壁酰残基的C-6羟基。OatA的结构和功能关系的信息是重要的，为未来的发展，可能作为抗病力剂的有效抑制剂。
The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens. Multi-drug resistance amongst important human pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and drug-resistant Streptococcus pneumoniae (DRSP), continues to challenge clinicians and threaten the lives of infected patients. Of the several approaches being taken to address this serious issue is the development of antagonists that render the bacterial infection more susceptible to the defensive enzymes and proteins of our innate immunity systems. One such target is the enzyme O-acetyltransferase A (OatA). This extracellular enzyme modifies the essential bacterial cell wall component peptidoglycan and thereby makes it resistant to the lytic action of lysozyme, our first line of defense against invading pathogens. In this study, we present the first biochemical and structural characterization of OatA. Using both the S. aureus and S. pneumoniae enzymes as model systems, we demonstrate that OatA has unique substrate specificities. We also show that the catalytic domain of OatA is a structural homolog of a well-studied superfamily of hydrolases. It uses a catalytic triad of Ser-His-Asp to transfer acetyl groups specifically to the C-6 hydroxyl group of muramoyl residues within peptidoglycan. This information on the structure and function relationship of OatA is important for the future development of effective inhibitors which may serve as antivirulence agents.
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DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
3.2
作者：
Burke, Thomas P.;Loukitcheva, Anastasia;Portnoy, Daniel A.
通讯作者：
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影响因子：
4.8
作者：
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通讯作者：
Chapot-Chartier, Marie-Pierre
影响因子：
3.6
作者：
Crisostomo, M. Ines;Vollmer, Waldemar;Tomasz, Alexander
通讯作者：
Tomasz, Alexander
影响因子：
6.4
作者：
Aubry, Camille;Goulard, Celine;Dussurget, Olivier
通讯作者：
Dussurget, Olivier