Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness,Biochemistry

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Alanine 501 Mutations in Penicillin-Binding Protein 2 from Neisseria gonorrhoeae: Structure, Mechanism, and Effects on Cephalosporin Resistance and Biological Fitness
Biochemistry ( IF 2.9 ) Pub Date : 2017-02-16 00:00:00 , DOI: 10.1021/acs.biochem.6b01030
Joshua Tomberg ₁ , Alena Fedarovich ₂ , Leah R. Vincent ₃ , Ann E. Jerse ₃ , Magnus Unemo ₄ , Christopher Davies ₂ , Robert A. Nicholas _{1,

5}

Affiliation

Resistance of Neisseria gonorrhoeae to expanded-spectrum cephalosporins such as ceftriaxone and cefixime has increased markedly in the past decade. The primary cephalosporin resistance determinant is a mutated penA gene, which encodes the essential peptidoglycan transpeptidase, penicillin-binding protein 2 (PBP2). Decreased susceptibility and resistance can be conferred by mosaic penA alleles containing upward of 60 amino acid changes relative to wild-type PBP2, or by nonmosaic alleles with relatively few mutations, the most important of which occurs at Ala501 located near the active site of PBP2. Recently, fully cefixime- and ceftriaxone-resistant clinical isolates that harbored a mosaic penA allele with an A501P mutation were identified. To examine the potential of mutations at Ala501 to increase resistance to expanded-spectrum cephalosporins, we randomized codon 501 in a mosaic penA allele and transformed N. gonorrhoeae to increased cefixime resistance. Interestingly, only five substitutions of Ala501 (A501V, A501T, A501P, A501R, and A501S) that increased resistance and preserved essential transpeptidase function were isolated. To understand their structural implications, these mutations were introduced into the nonmosaic PBP2-6140CT, which contains four C-terminal mutations present in PBP2 from the penicillin-resistant strain FA6140. The crystal structure of PBP2-6140CT-A501T was determined and revealed ordering of a loop near the active site and a new hydrogen bond involving Thr501 that connects the loop and the SxxK conserved active site motif. The structure suggests that increased rigidity in the active site region is a mechanism for cephalosporin resistance mediated by Ala501 mutations in PBP2.

中文翻译：

淋病奈瑟菌青霉素结合蛋白2的丙氨酸501突变：结构，机理，以及对头孢菌素抗性和生物适应性的影响。

在过去的十年中，淋病奈瑟氏球菌对头孢曲松和头孢克肟等广谱头孢菌素的耐药性显着增加。主要的头孢菌素抗性决定簇是一个突变的penA基因，它编码必需的肽聚糖转肽酶，青霉素结合蛋白2（PBP2）。可以通过相对于野生型PBP2含有60个以上氨基酸变化的镶嵌penA等位基因，或具有相对较少突变的非镶嵌等位基因，来降低敏感性和耐药性，其中最重要的突变发生在位于PBP2活性位点附近的Ala501。最近，带有花叶笔的完全对头孢克肟和头孢曲松耐药的临床分离株鉴定出具有A501P突变的等位基因。为了检查Ala501处突变的可能性，以增加其对广谱头孢菌素的抗性，我们将镶嵌penA等位基因中的501密码子随机化并转化了淋病奈瑟氏球菌。对头孢克肟的耐药性增加。有趣的是，仅分离了五个Ala501取代（A501V，A501T，A501P，A501R和A501S），这些取代增加了抗性并保留了必需的转肽酶功能。为了解其结构含义，将这些突变引入了非马赛克PBP2-6140CT中，该突变包含来自青霉素耐药菌株FA6140的PBP2中存在的四个C端突变。确定了PBP2-6140CT-A501T的晶体结构，并揭示了活性位点附近的环的顺序以及涉及连接环和SxxK保守活性位点基序的Thr501的新氢键。该结构表明，活性位点区域刚性的增加是由PBP2中Ala501突变介导的头孢菌素耐药性的机制。

更新日期：2017-02-16

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