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.
Highlights
VanX belongs to the vanA gene cluster, which confers vancomycin resistance by protecting the bacterial cell wall
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
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
Summary
VanX belongs to the vanA gene cluster, which confers vancomycin resistance by protecting the bacterial cell wall. 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. 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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