A Streptococcus aquaporin acts as peroxiporin for efflux of cellular hydrogen peroxide and alleviation of oxidative stress

Abstract

Edited by Ursula JakobAquaporins (AQPs) are transmembrane proteins widely distributed in various organisms, and they facilitate bidirectional diffusion of water and uncharged solutes. The catalase-negative bacterium Streptococcus oligofermentans produces the highest H 2 O 2 levels reported to date, which has to be exported to avoid oxidative stress. Here, we report that a S. oligofermentans aquaporin functions as a peroxiporin facilitating bidirectional transmembrane H 2 O 2 transport. Knockout of this aquaporin homolog, So-AqpA, reduced H 2 O 2 export by ϳ50% and increased endogenous H 2 O 2 retention, as indicated by the cellular H 2 O 2 reporter HyPer. Heterologous expression of So-aqpA accelerated exogenous H 2 O 2 influx into Saccharomyces cerevisiae and Escherichia coli cells, indicating that So-AqpA acts as an H 2 O 2transferring aquaporin. Alanine substitution revealed Phe-40 as a key residue for So-AqpA-mediated H 2 O 2 transport. Northern blotting, qPCR, and luciferase reporter assays disclosed that H 2 O 2 induces a >10-fold expression of So-aqpA. Super-resolution imaging showed that H 2 O 2 treatment increases So-AqpA protein molecules per cell by 1.6-to 3-fold. Inactivation of two redox-regulatory transcriptional repressors, PerR and MntR, reduced H 2 O 2 -induced So-aqpA expression to 1.8-and 4-fold, respectively. Electrophoretic mobility shift assays determined that MntR, but not PerR, binds to the So-aqpA promoter, indicating that MntR directly regulates H 2 O 2 -induced So-aqpA expression. Importantly, So-aqpA deletion decreased oxic growth and intraspecies competition and diminished the competitive advantages of S. oligofermentans over the caries pathogen Streptococcus mutans. Of note, So-aqpA orthologs with the functionally important Phe-40 are present in all streptococci. Our work has uncovered an intrinsic, H 2 O 2 -inducible bacterial peroxiporin that has a key physiological role in H 2 O 2 detoxification in S. oligofermentans.

Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

“…Given that streptococci accumulate endogenous H 2 O 2 , we used the HyPer fluorescent protein to estimate intracellular H 2 O 2 levels ( 32 ) and compared them with those excreted into the culture. The wild-type (WT) HyPer reporter strain of S. oligofermentans , WT-HyPer ( 33 ), was statically grown in 10, 20, 30, and 40 ml of brain heart infusion (BHI) broth in 100-ml flasks, which built an initial O 2 supply gradient. The growth profiles and the H 2 O 2 amounts in the cultures were measured.…”

“…Next, the mid-exponential-phase cells of the WT-HyPer strain from each volume of cultures were visualized under a confocal laser scanning microscope (Leica model TCS SP8), and the HyPer fluorescence intensities were measured as described in Materials and Methods. The Δ pox -HyPer mutant, the pox deletion mutant carrying the HyPer gene ( 33 ), was included as a control from which H 2 O 2 was absent. Figure 1B and C show that the HyPer fluorescence intensities were inversely proportional to the culture volumes but directly proportional to the H 2 O 2 concentrations in the cultures, with a good linear regression ( R 2 = 0.8745) ( Fig.…”

Connective tissue fibroblasts from highly regenerative mammals are refractory to ROS-induced cellular senescence

“…7b). These conditions allowed us to monitor a single wavelength to quantify intracellular H 2 O 2 levels 39–42 . This data showed peak fluorescence levels were not different across species 30 min after H 2 O 2 treatment (Fig.…”

“…Intracellular H 2 O 2 levels are usually quantified by calculating the ratio of two excitation wavelengths, 420 nm and 500 nm. However, it is appropriate to use single wavelength monitoring if cell movement is minimal and sensor expression levels are similar across cells as occurs in our experimental design 39–42 . For experiments using HyPer, equal numbers of cells were seeded into 24-well glass bottom plates until they reached 70–80% confluency.…”

“…The fluorescent intensity of HyPer was measured using a FITC channel with excitation and emission wavelength (ex/em) at 495/520 nm. Although the HyPer probe is often detected by calculating the ratio of two excitation wavelengths (420 and 500 nm), we monitored a single wavelength because there was negligible movement of fibroblasts during live imaging and we controlled for sensor transfection from all four species 39–42 . To account for potential variation in sensor transfection, we analyzed cells (≥20 cells/cell line) exhibiting similar baseline expression levels of HyPer across all four species.…”