Metallo-β-lactamases (MBLs) are a class of proteins contributing to the ongoing but often unrecognized antibiotic resistance crisis, in which many current and most past antibiotics are less effective than when first introduced. These proteins protect bacteria by destroying manmade antibiotics, and have spread worldwide in part due to the selective pressure of antibiotic overprescription. To develop new compounds to inhibit these proteins and protect from resistant “superbugs,” researchers characterize groups of MBLs to learn why and how they are evolving, and hopefully identify weaknesses. Last year, I was involved in the collaborative examination of the MBL VIM-2 and its single-mutation variant, VIM-20. While identifying these proteins as noteworthy, additional proteins within the Verona Integron-encode MBL (VIM) family were considered but ignored due to difficulty in harvesting them. This year, successful sample preparation allowed for the examination of metal-binding (ion-coupled plasma optical emission spectroscopy; native mass spectrometry), kinetics (UV-Vis spectroscopy), and thermostability (differential scanning fluorimetry) of VIM-4 and several of its single-mutation variants (VIM-19, VIM-28, and VIM-37) to compare with the previous study. Metal-binding studies variably showed a second zinc (used by VIM and other MBLs to degrade antibiotics) binding site on VIM-4 and VIM-37. Kinetic studies, using the antibiotic meropenem, indicated increased efficiency and speed of antibiotic degradation of the variants compared to VIM-4 itself, a relationship unlike that between VIM-20 and VIM-2. Finally, although VIM-20 showed increased thermostability versus VIM-2 especially at physiologically relevant conditions, VIM-4 and its variants proved too unstable to test successfully, while also demonstrating rapid activity decreases at room temperature. Although the kinetic activity is the tentatively assigned driving force for VIM-4 subfamily variability, further investigations into kinetic behavior with other antibiotics and conditions, optimization of thermostability experimental procedure, and structure analysis were planned and will likely recommence following the current national healthcare situation.
Author: Robert L. Kimble
Faculty Advisor: Dr. Michael W. Crowder, PhD, Dept. of Chemistry & Biochemistry
Graduate Students Advisors (and others): Dr. Zishuo Cheng (Post-doctoral staff), Caitlyn Thomas, Kundi Yang
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