Biochemical and Biophysical Characterization of an Unexpected Bacteriolytic Activity of VanX,a Member of the Vancomycin-resistance vanA Gene Cluster |
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Authors: | Shihori Sohya Tetsuya Kamioka Chisako Fujita Tei Maki Yoshihiro Ohta Yutaka Kuroda |
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Institution: | From the ‡Department of Biotechnology and Life Science, Graduate School of Engineering, and ;§Research Center for Science and Technology, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi, Tokyo, 184-8588 Japan, and ;¶JEOL Ltd., EM Business Unit, 3-1-2 Musashino, Akishima, Tokyo 196-8558, Japan |
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Abstract: | VanX is a d-alanyl-d-alanine (d-Ala–d-Ala) dipeptidase encoded in the vancomycin-resistance vanA gene cluster. Here we report that strong bacteriolysis occurred when isolated VanX was expressed in Escherichia coli at temperatures lower than 30 °C, which was unexpected because the vanA operon confers vancomycin resistance by protecting the cell wall. Therefore, we monitored cell lysis by measuring sample turbidity with absorbance at 590 nm and VanX expression using SDS-PAGE. No cell lysis was observed when VanX was expressed, even in large quantities, in the cell inclusion bodies at 37 °C, suggesting that a natively folded VanX is required for lysis. In addition, VanX mutants with suppressed dipeptidase activity did not lyse E. coli cells, confirming that bacteriolysis originated from the dipeptidase activity of VanX. We also observed shape changes in E. coli cells undergoing VanX-mediated lysis with optical microscopy and classified these changes into three classes: bursting, deformation, and leaking fluid. Optical microscopic image analysis fully corroborated our interpretation of the turbidity changes in the samples. From a practical perspective, the finding that VanX expressed in isolation induces cell lysis suggests that inhibitors of VanA and VanH that act downstream from VanX could provide a new class of therapeutic chemicals against bacteria expressing the vancomycin-resistance gene cluster. |
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Keywords: | Antibiotic Resistance Bacterial Pathogenesis Cell Wall Peptidase Peptidoglycan Murein |
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