Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

Abstract

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M−1s−1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

Oxidative Stress in Age-Related Neurodegenerative Diseases: An Overview of Recent Tools and Findings

“…All tools, along with their application method and their key features, reported in this section are summarized in Table 1 to assist the reader in locating the most suitable tool for their purpose. Hypobromous acid (HOBr) RhSN-mito a Ratiometric probe Selective over HOCl [53] All hypohalous acids (HOX) Hypocrates b First biosensor specific for HOX, ratiometric imaging [54] Superoxide anion (O2…”

“…While hypohalous acids react rapidly with cysteines, selectivity is hard to be achieved as cysteines are susceptible to oxidation by many other ROS. In order to develop a HOX-selective biosensor, Belousov and co-workers utilized the E. coli-derived transcription repressor NemR [54], a protein that had previously been shown to contain a certain cysteine (Cys106) prone to oxidation by chlorine species [78]. The team therefore developed the so-called Hypocrates sensor by integrating a YFP protein between the DNA-binding and the sensory domain of NemR, the latter containing Cys106 (Figure 3D).…”

Hypothiocyanite and host–microbe interactions

“…The rclBC genes are generally conserved only in enterobacteria (Derke et al, 2020). RclR also responds strongly to reactive chlorine compounds (Parker et al, 2013), and since the HOCl-responsive E. coli transcription factor NemR also appears to detect HOSCN (Kostyuk et al, 2022), we suspect that many cysteine-dependent HOCl-sensitive regulators in bacteria (da Cruz Nizer et al, 2020) may also respond to HOSCN, but this remains to be determined. A homolog of RclC has recently been characterized in uropathogenic E. coli (UPEC) and has been implicated in aiding with membrane stability when cells are exposed to HOCl (Sultana et al, 2022), but the mechanism by which it does so remains unknown.…”

“…The presence of HOCl in biological samples can be inferred from the detection of HOCl‐specific biomarkers (e.g., 3‐chlorotyrosine or glutathione sulfonamide) (Harwood et al, 2008; Kettle et al, 2014), but no specific biomarkers produced by HOSCN oxidation of biological molecules are known. A recently described fluorescent biosensor based on the HOCl‐sensing transcription factor NemR is reported to respond to HOSCN (Kostyuk et al, 2022), but it also responds to HOCl, so similarly lacks specificity.…”

Guidelines for measuring reactive oxygen species and oxidative damage in cells and in vivo

“…More specific fluorescent probes for reactive halogen species have been reported and some are commercially available 52 . A genetically encoded probe for reactive halogen species has been developed, enabling dynamic monitoring of these species both in cell culture and in vivo 53 .…”