Papp-Wallace Krisztina M, Winkler Marisa L, Taracila Magdalena A, Bonomo Robert A
Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
Research Service, Louis Stokes Cleveland Department of Veterans Affairs, Cleveland, Ohio, USA Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio, USA.
Antimicrob Agents Chemother. 2015 Jul;59(7):3710-7. doi: 10.1128/AAC.04406-14. Epub 2015 Feb 9.
KPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling. Escherichia coli DH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; the k2/K ratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M(-1) s(-1) to 1.2 M(-1) s(-1)). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent against E. coli DH10B strains possessing all of these variants.
KPC-2是全球最普遍的A类碳青霉烯酶。此前研究表明，KPC-2能够水解β-内酰胺酶抑制剂克拉维酸、舒巴坦和他唑巴坦。此外，KPC-2β-内酰胺酶中氨基酸位置R220的替换增强了对克拉维酸的耐药性。一种新型桥连二氮杂双环辛烷（DBO）非β-内酰胺β-内酰胺酶抑制剂阿维巴坦，已被证明可使KPC-2β-内酰胺酶失活。为了更好地理解阿维巴坦抑制KPC-2的机制基础，我们测试了氨苄西林-阿维巴坦和头孢他啶-阿维巴坦对KPC-2β-内酰胺酶工程变体的效力，这些变体在重要位点（即安布勒编号69、130、234、220和276）有单个氨基酸替换，此前已证明这些位点在TEM和SHVβ-内酰胺酶中可赋予抑制剂耐药性。为此，我们进行了药敏试验、生化分析和分子建模。携带替换为S130G、K234R和R220M的KPC-2β-内酰胺酶变体的大肠杆菌DH10B，仅对氨苄西林-阿维巴坦组合的最低抑菌浓度（MIC）升高（例如，分别为512、64和32mg/L，而野生型KPC-2的MIC为2至8mg/L）。稳态动力学表明，KPC-2的S130G变体抵抗阿维巴坦的失活；S130G变体的k2/K比值比野生型酶的比值显著降低4个对数（从21,580M-1s-1降至1.2M-1s-1）。分子建模和分子动力学模拟表明，KPC-2的S130G变体中K73的流动性及其激活S70的能力（即作为通用碱发挥作用）可能受损，从而解释了酰化减慢的原因。此外，我们还提出，在S130G变体中，阿维巴坦硫酸根氮的质子化可能减慢，因为S130可能是质子供体，另一个残基（可能是K234）必须进行补偿。我们的研究结果表明，残基S130以及K234和R220对阿维巴坦使KPC-2失活的机制有显著贡献。幸运的是，临床上KPC的S130G、K234R和R220M变体的出现不应导致头孢他啶-阿维巴坦治疗失败，因为头孢他啶对携带所有这些变体的大肠杆菌DH10B菌株有效。
