A Fluorescent Probe to Detect Quick Disulfide Reductase Activity in Bacteria.

Ying Zhao,Jun Lu,Wenjun Qian,Shuang Liu,Xin Zuo,Xuewen Tang

doi:10.3390/antiox11020377

Abstract

The Trx and Grx systems, two disulfide reductase systems, play critical roles in various cell activities. There are great differences between the thiol redox systems in prokaryotes and mammals. Though fluorescent probes have been widely used to detect these systems in mammalian cells. Very few methods are available to detect rapid changes in the redox systems of prokaryotes. Here we investigated whether Fast-TRFS, a disulfide-containing fluorescent probe utilized in analysis of mammalian thioredoxin reductase, could be used to detect cellular disulfide reducibility in bacteria. Fast-TRFS exhibited good substrate qualities for both bacterial thioredoxin and GSH-glutaredoxin systems in vitro, with Trx system having higher reaction rate. Moreover, the Fast-TRFS was used to detect the disulfide reductase activity in various bacteria and redox-related gene null E. coli. Some glutaredoxin-deficient bacteria had stronger fast disulfide reducibility. The Trx system was shown to be the predominant disulfide reductase for fast disulfide reduction rather than the Grx system. These results demonstrated that Fast-TRFS is a viable probe to detect thiol-dependent disulfide reductases in bacteria. It also indicated that cellular disulfide reduction could be classified into fast and slow reaction, which are predominantly catalyzed by E. coli Trx and Grx system, respectively.

Highlights

Oxidative stress is one of the most common and challenging conditions for bacterial homeostasis and survival [1,2]
Fast-TRFS can react with mammalian thioredoxin reductase (TrxR) within one step, which is the disulfide bond of the probe reduced by TrxR and yielding blue fluorescence
These results indicated that the two probes, Fast-TRFS and Naph-EA-mal, could enter bacterial cells and they can be used in the analysis of cellular fast disulfide reducibility bacterial cells and they can be used in the analysis of cellular fast disulfide reducibility and and total total thiols

Summary

Introduction

Oxidative stress is one of the most common and challenging conditions for bacterial homeostasis and survival [1,2]. Prokaryotic microorganisms evolved various antioxidant systems to mediate the challenge [3,4]. Thiol-dependent redox systems play a fundamental role in maintaining the redox balance in bacteria [5,6]. Most Gram-negative bacteria have two primary thiol-dependent antioxidant pathways called thioredoxin (Trx) and glutaredoxin (Grx) systems that use nicotinamide adenine dinucleotide phosphate (NADPH). To reduce intracellular disulfides [6,7,8]. In the Trx system, electrons are transferred from. NADPH to thioredoxin reductase (TrxR), to Trx, while in the Grx system, it is glutathione reductase (GR) and glutathione (GSH), to glutaredoxins.

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