To understand the evolution of Verona integron-encoded metallo-β-lactamase (VIM) genes (bla VIM) and their clinical impact, microbiological, biochemical, and structural studies were conducted. Forty-five clinically derived VIM variants engineered in a uniform background and expressed in Escherichia coli afforded increased resistance toward all tested antibiotics; the variants belonging to the VIM-1-like and VIM-4-like families exhibited higher MICs toward five out of six antibiotics than did variants belonging to the widely distributed and clinically important VIM-2-like family. Generally, maximal MIC increases were observed when cephalothin and imipenem were tested. Additionally, MIC determinations under conditions with low zinc availability suggested that some VIM variants are also evolving to overcome zinc deprivation. The most profound increase in resistance was observed in VIM-2-like variants (e.g., VIM-20 H229R) at low zinc availability. Biochemical analyses reveal that VIM-2 and VIM-20 exhibited similar metal binding properties and steady-state kinetic parameters under the conditions tested. Crystal structures of VIM-20 in the reduced and oxidized forms at 1.25 Å and 1.37 Å resolution, respectively, show that Arg229 forms an additional salt bridge with Glu171. Differential scanning fluorimetry of purified proteins and immunoblots of periplasmic extracts revealed that this difference increases thermostability and resistance to proteolytic degradation when zinc availability is low. Therefore, zinc scarcity appears to be a selective pressure driving the evolution of multiple metallo-β-lactamase families, although compensating mutations use different mechanisms to enhance resistance.IMPORTANCE Antibiotic resistance is a growing clinical threat. One of the most serious areas of concern is the ability of some bacteria to degrade carbapenems, drugs that are often reserved as last-resort antibiotics. Resistance to carbapenems can be conferred by a large group of related enzymes called metallo-β-lactamases that rely on zinc ions for function and for overall stability. Here, we studied an extensive panel of 45 different metallo-β-lactamases from a subfamily called VIM to discover what changes are emerging as resistance evolves in clinical settings. Enhanced resistance to some antibiotics was observed. We also found that at least one VIM variant developed a new ability to remain more stable under conditions where zinc availability is limited, and we determined the origin of this stability in atomic detail. These results suggest that zinc scarcity helps drive the evolution of this resistance determinant.

中文翻译：

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在低锌条件下，VIM-20中的单个盐桥可提高蛋白质稳定性和抗生素抗性。

为了了解维罗纳整合素编码的金属β-内酰胺酶（VIM）基因（bla VIM）的进化及其临床影响，进行了微生物学，生化和结构研究。在统一背景下工程化并在大肠杆菌中表达的45种临床衍生的VIM变体对所有测试抗生素的耐药性有所提高。与属于广泛分布和临床上重要的VIM-2类家族的变体相比，属于VIM-1类和VIM-4类的变体对六种抗生素中的五种表现出更高的MIC。通常，当测试头孢菌素和亚胺培南时，观察到最大的MIC增加。此外，在低锌可用性条件下的MIC测定表明，一些VIM变体也在不断发展，以克服锌缺乏的问题。在锌利用率低的情况下，在类似VIM-2的变体（例如VIM-20 H229R）中观察到了最大的电阻增加。生化分析表明，在测试条件下，VIM-2和VIM-20表现出相似的金属结合性能和稳态动力学参数。分别以1.25Å和1.37Å分辨率还原和氧化的VIM-20晶体结构表明Arg229与Glu171形成了另一个盐桥。纯化蛋白和周质提取物的免疫印迹的差示扫描荧光法显示，当锌的利用率低时，这种差异增加了热稳定性和对蛋白水解降解的抵抗力。因此，锌缺乏似乎是驱动多个金属β-内酰胺酶家族进化的选择性压力，尽管补偿性突变使用不同的机制来增强耐药性。重要提示抗生素耐药性是日益增长的临床威胁。最令人关注的领域之一是某些细菌降解碳青霉烯的能力，碳青霉烯通常被保留为最后使用的抗生素。对碳青霉烯类的抗药性可以由一大批称为金属β-内酰胺酶的相关酶赋予，这些酶依赖锌离子来发挥功能和整体稳定性。在这里，我们研究了来自称为VIM的亚科的45种不同的金属β-内酰胺酶的广泛研究小组，以发现随着耐药性在临床环境中的发展，正在发生什么变化。观察到对某些抗生素的抵抗力增强。我们还发现，至少有一种VIM变体开发出一种新功能，可以在锌供应有限的条件下保持更稳定，我们从原子细节上确定了这种稳定性的起源。这些结果表明锌缺乏有助于推动该抗性决定簇的进化。