Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles.

Nathan J Alves,Scott A Walper,Kendrick B Turner,Igor L Medintz

doi:10.1038/srep24866

Abstract

Bacteria possess innate machinery to transport extracellular cargo between cells as well as package virulence factors to infect host cells by secreting outer membrane vesicles (OMVs) that contain small molecules, proteins, and genetic material. These robust proteoliposomes have evolved naturally to be resistant to degradation and provide a supportive environment to extend the activity of encapsulated cargo. In this study, we sought to exploit bacterial OMV formation to package and maintain the activity of an enzyme, phosphotriesterase (PTE), under challenging storage conditions encountered for real world applications. Here we show that OMV packaged PTE maintains activity over free PTE when subjected to elevated temperatures (>100-fold more activity after 14 days at 37 °C), iterative freeze-thaw cycles (3.4-fold post four-cycles), and lyophilization (43-fold). We also demonstrate how lyophilized OMV packaged PTE can be utilized as a cell free reagent for long term environmental remediation of pesticide/chemical warfare contaminated areas.

Highlights

Promiscuous enzyme capable of hydrolyzing a wide variety of organophosphate compounds including V and G type chemical warfare agents as well as toxic pesticides such as paraoxon, its primary substrate[24,25,26]
A biorthogonal conjugation strategy was developed to create a synthetic linkage between an enzyme of interest (PTE) and a highly expressed transmembrane porin protein (OmpA) known to be abundant in outer membrane vesicle (OMV)
It was shown previously that when co-transformed with PTE-SC, both the N- and C- terminal OmpA-SpyTag fusions exhibited hyper-vesiculation, improved production of PTE, and increased packaging efficiency of PTE within the OMVs. These characteristics make them desirable constructs for testing the stability of enzyme packaged OMVs as well as viable environmental remediation reagents[22]

Summary

Introduction

Promiscuous enzyme capable of hydrolyzing a wide variety of organophosphate compounds including V and G type chemical warfare agents as well as toxic pesticides such as paraoxon, its primary substrate[24,25,26]. We utilized a split protein SpyCatcher/SpyTag bioconjugation system to create a synthetic linkage between PTE and an abundant membrane protein, OmpA, to facilitate loading of the OMV27,28. A complete analysis of the bacterial vesiculation, packaging efficiency, overall PTE production, and enzymatic activity of the packaged PTE was carried out previously to validate the constructs[22]. We demonstrate how packaging PTE within an OMV utilizing these well characterized constructs greatly improves enzymatic activity across a range of conditions including: long-term storage, exposure to elevated temperatures, freeze-thaw cycles, and lyophilization allowing for enhanced potential for PTE use in real world environmental remediation applications (Fig. 1)

Methods

Results

Conclusion