Green fluorescent proteins induce oxidative stress in cells: A worrisome new wrinkle in the application of the GFP reporter system to biological systems?

Kalyanaraman, Balaraman; Zielonka, Jacek

doi:10.1016/j.redox.2017.03.019

Cited by 23 publications

(17 citation statements)

References 16 publications

(16 reference statements)

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“…When we prevented queue formation using chloramphenicol, adding IPTG did not affect survival of ampicillin. While CFP production alone slightly increased survival for ciprofloxacin, we suspect that high production of CFP with no apparent method of removal (besides cell division; minimal degradation) causes cell stress and affects survival, especially since high levels of fluorescent proteins can cause oxidative stress 45,46 , which is known to increase persistence [47][48][49] . However, because CFP- LAA is removed via degradation (indicated by lower fluorescence than CFP-untagged), the effects seen via overexpression of CFP should be less prominent during CFP-LAA overexpression.…”

Section: Discussionmentioning

confidence: 93%

Proteolytic queues at ClpXP increase antibiotic tolerance

Deter

Abualrahi

Jadhav

et al. 2019

Preprint

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Antibiotic tolerance is a widespread phenomenon that renders antibiotic treatments less effective and facilitates antibiotic resistance. Here we explore the role of proteases in antibiotic tolerance, short-term population survival of antibiotics, using queueing theory (i.e. the study of waiting lines), computational models, and a synthetic biology approach. Proteases are key cellular components that degrade proteins and play an important role in a multi-drug tolerant subpopulation of cells, called persisters. We found that queueing at the protease ClpXP increases antibiotic tolerance ~80 and ~60 fold in an E. coli population treated with ampicillin and ciprofloxacin, respectively. There does not appear to be an effect on antibiotic persistence, which we distinguish from tolerance based on population decay. These results demonstrate that proteolytic queueing is a practical method to probe bacterial tolerance and related genes, while limiting the unintended consequences frequently caused by gene knockout and overexpression.

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Section: Discussionmentioning

confidence: 93%

Proteolytic queues at ClpXP increase antibiotic tolerance

Deter

Abualrahi

Jadhav

et al. 2019

Preprint

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“…Furthermore, some genetically-encoded fluorescent proteins directly generate • O 2¯a fter excitation. [85][86][87][88][89][90][91] The main focus of our review article is on electrochemical and electron spin resonance (ESR) spectroscopic methods for determination of H 2 O 2 . Both allow precise [H 2 O 2 ] quantification also in a cellular context.…”

Section: Redox Chemistry Of Hydrogen Peroxidementioning

confidence: 99%

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Gulaboski

Mirčeski

Kappl

et al. 2019

J. Electrochem. Soc.

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Reactive oxygen species (ROS) exhibit different spatial and temporal distributions as well as concentrations in-and outside the cell, thereby functioning as signaling or pathogen-destroying molecules. Especially the ROS H 2 O 2 is important for the patho/physiological status of an organism. Electrochemistry (EM) and electron spin resonance (ESR)-based techniques allow quantification of H 2 O 2 in artificial and living systems, coping a concentration range from low nM up to mM. Working electrodes for EM are optimized by diverse modifications and, additionally, redox mediators are used. Ultramicroelectrodes allow scanning of single cells to spatially resolve and quantify extracellular H 2 O 2 in real-time. With ESR spectroscopy, • O 2¯, but not H 2 O 2 , can be directly determined by spin probes in-and outside of cells in suspensions. Monitoring H 2 O 2 requires formation of intermediate radicals, detectable with spin probes. Low μM [H 2 O 2 ] can thus be assessed specifically. Using suitable spin traps, in-vivo ESR and immuno-spin trapping can visualize different radicals at their respective production sites in small animals, organs and tissues. Here, the redox reaction cascades may interfere with cell metabolism. Optimization of all methods established for H 2 O 2 determination would be favorable to finally combine them for mutual validation. Thus, a deeper insight into cellular ROS metabolism can be obtained.

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“…HeLa cells stably expressing GFP had increased rates of oxidant production, which led to changes in gene expression in oxidant responsive pathways [58]. Overall, mere expression of GFP in cells may impact the redox status [58, 59]. GFP also generates oxidants in response to illumination.…”

Section: Green Fluorescent Protein (Gfp)mentioning

confidence: 99%

Light-induced oxidant production by fluorescent proteins

Trewin

Berry

Wei

et al. 2018

Free Radical Biology and Medicine

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Oxidants play an important role in the cell and are involved in many redox processes. Oxidant concentrations are maintained through coordinated production and removal systems. The dysregulation of oxidant homeostasis is a hallmark of many disease pathologies. The local oxidant microdomain is crucial for the initiation of many redox signaling events; however, methods to control oxidant product are limited. Some fluorescent proteins, including GFP, TagRFP, KillerRed, miniSOG, and their derivatives, generate oxidants in response to light. These genetically-encoded photosensitizers produce singlet oxygen and superoxide upon illumination and offer spatial and temporal control over oxidant production. In this review, we will examine the photosensitization properties of fluorescent proteins and their application to redox biology. Emerging concepts of selective oxidant species production via photosensitization and the impact of light on biological systems are discussed.

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Green fluorescent proteins induce oxidative stress in cells: A worrisome new wrinkle in the application of the GFP reporter system to biological systems?

Cited by 23 publications

References 16 publications

Proteolytic queues at ClpXP increase antibiotic tolerance

Proteolytic queues at ClpXP increase antibiotic tolerance

Review—Quantification of Hydrogen Peroxide by Electrochemical Methods and Electron Spin Resonance Spectroscopy

Light-induced oxidant production by fluorescent proteins

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