Abstract
Aquaporins (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 H2O2 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 H2O2 transport. Knockout of this aquaporin homolog, So-AqpA, reduced H2O2 export by ∼50% and increased endogenous H2O2 retention, as indicated by the cellular H2O2 reporter HyPer. Heterologous expression of So-aqpA accelerated exogenous H2O2 influx into Saccharomyces cerevisiae and Escherichia coli cells, indicating that So-AqpA acts as an H2O2-transferring aquaporin. Alanine substitution revealed Phe-40 as a key residue for So-AqpA-mediated H2O2 transport. Northern blotting, qPCR, and luciferase reporter assays disclosed that H2O2 induces a >10-fold expression of So-aqpA Super-resolution imaging showed that H2O2 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 H2O2-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 H2O2-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, H2O2-inducible bacterial peroxiporin that has a key physiological role in H2O2 detoxification in S. oligofermentans.
Highlights
Aquaporins (AQPs) are transmembrane proteins widely distributed in various organisms, and they facilitate bidirectional diffusion of water and uncharged solutes
The S. oligofermentans genome carries three major intrinsic protein (MIP) family homologous genes: I872_01445 encodes an aquaporin, so was designated as So-aqpA and the encoded protein designated as SoAqpA; I872_09070 and I872_03455 encode glycerol uptake facilitator proteins and are designated as So-aqpB and So-aqpC, respectively, and the encoded proteins were designated as SoAqpB and So-AqpC
So-AqpA is related to the E. coli aquaporin Z (b0875), whereas the two aquaglyceroporins (So-AqpB and So-AqpC) are related to the E. coli aquaglyceroporin GlpF (b3927) and the human aquaporin AQP3 [360] (Fig. 1A)
Summary
Because of the limited studies on prokaryotic AQPs [2, 20, 21], their functions are largely unknown, such as whether they function to facilitate H2O2 excretion and whether this action has physiological significance. Based on the rate of H2O2 flux, Seaver and Imlay [22] found that a H2O2 transmembrane concentration gradient exists in Escherichia coli. This suggests that H2O2 permeability across the cellular membrane is limited, and the bacterial aquaporins may function in facilitating H2O2 diffusion. Streptococci carry genes that encode two clades of aquaporins, AQPs and GLPs. Interestingly, our previous microarray analysis found increased expression of the AQP genes in H2O2-treated Streptococcus oligofermentans, a bacterium so far known to produce and tolerate the highest levels of H2O2 [23], implying that the AQPs might play a role in H2O2 excretion. This work reports for the first time the physiological roles of a bacterial peroxiporin, which could be a potential target for suppression of streptococci, especially the pathogenic species
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