A Novel Aquaporin Subfamily Imports Oxygen and Contributes to Pneumococcal Virulence by Controlling the Production and Release of Virulence Factors.

Aquaporins, integral membrane proteins widely distributed in organisms, facilitate the transport of water, glycerol, and other small uncharged solutes across cellular membranes and play important physiological roles in eukaryotes. However, characterizations and physiological functions of the prokaryotic aquaporins remain largely unknown. Here, we report that Streptococcus pneumoniae (pneumococcus) AqpC (Pn-AqpC), representing a new aquaporin subfamily possessing a distinct substrate-selective channel, functions as an oxygen porin by facilitating oxygen movement across the cell membrane and contributes significantly to pneumococcal virulence. The use of a phosphorescent oxygen probe showed that Pn-AqpC facilitates oxygen permeation into pneumococcal and Pn-AqpC-expressing yeast cells. Reconstituting Pn-AqpC into liposomes prepared with pneumococcal and Escherichia coli cellular membranes further verified that Pn-AqpC transports O2 but not water or glycerol. Alanine substitution showed that Pro232 in the substrate channel is key for Pn-AqpC in O2 transport. The deletion of Pn-aqpC significantly reduced H2O2 production and resistance to H2O2 and NO of pneumococci, whereas low-H2O2 treatment helped the ΔPn-aqpC mutant resist higher levels of H2O2 and even NO, indicating that Pn-AqpC-facilitated O2 permeation contributes to pneumococcal resistance to H2O2 and NO. Remarkably, the lack of Pn-aqpC alleviated cell autolysis, thus reducing pneumolysin (Ply) release and decreasing the hemolysis of pneumococci. Accordingly, the ΔPn-aqpC mutant markedly reduced survival in macrophages, decreased damage to macrophages, and significantly reduced lethality in mice. Therefore, the oxygen porin Pn-AqpC, through modulating H2O2 production and pneumolysin release, the two major pneumococcal virulence factors, controls the virulence of pneumococcus. Pn-AqpC orthologs are widely distributed in various pneumococcal serotypes, highlighting that the oxygen porin is important for pneumococcal pathogenicity. IMPORTANCE Pneumococcus is the leading cause of community-acquired pneumonia, bacteremia, and meningitis. This work reports that a novel aquaporin subfamily represented by pneumococcal Pn-AqpC functions as an oxygen porin facilitating O2 influx into cells. Importantly, by mediating O2 influx, Pn-AqpC controls the production and release of H2O2 and Ply, the two major pneumococcal virulence factors. Moreover, by enhancing endogenous H2O2 production, Pn-AqpC significantly increases pneumococcal resistance to H2O2 and even NO, the major bactericidal chemical produced by macrophages. Consequently, the deletion of Pn-aqpC markedly decreased pneumococcal survival in macrophages and reduced damage to macrophages. Accordingly, the ΔPn-aqpC mutant displays significantly attenuated virulence in a murine pneumonia model. Given that Pn-AqpC orthologs are widely distributed in all pneumococcal serotypes, this new subfamily of aquaporins is identified as novel virulence-related proteins.

INTRODUCTION

Streptococcus pneumoniae (pneumococcus) is a major cause of community-acquired pneumonia, bacteremia, and meningitis (1, 2). It relies on multiple virulence factors, capsular polysaccharides (CPSs), pneumolysin (Ply), and H2O2, to transmit and escape the host’s innate immune system (1, 3–5). Distinguishingly, pneumococcus produces as well as resists high concentrations of H2O2 (6, 7). The ability to resist oxidative stress enables pneumococcus to survive against killing by host phagocytes (8, 9); particularly, intramacrophage survival is key to effective septic infection by pneumococcus (10). However, excess endogenous H2O2 also imposes oxidative stress on pneumococcus (11); therefore, effective efflux of H2O2 would be a way for detoxification. Recently, an aquaporin of Streptococcus oligofermentans, a close relative of pneumococcus, was reported to facilitate the efflux of cellular H2O2 (12). This provides a clue that pneumococcus may also employ aquaporins not only for the efflux of H2O2 for detoxification but also for virulence.

Aquaporins are integral membrane proteins found in diverse organisms (13–15). They facilitate the diffusion of water, glycerol, H2O2, ammonia, and other small uncharged solutes across cellular membranes and play important roles in physiological activities and diseases in eukaryotes (13, 15–19). Aquaporins are categorized into three major phylogenetic subfamilies: the classical water-transporting aquaporins (AQPs), the glycerol-transporting aquaglyceroporins (AQGPs), and the AQP supergene channel superaquaporins (SAQPs) (14). The three aquaporin subfamilies differ in their amino acid compositions of the aromatic/arginine constriction region (ar/R region), also known as the selective filter (20). However, only a few prokaryotic aquaporins have been studied regarding their physiological functions (21, 22).

Unexpectedly, we found that pneumococcus and some streptococcal pathogenic species encode a distinct aquaporin ortholog distantly related to the Escherichia coli glycerol facilitator GlpF, thus possibly representing a new aquaporin subfamily, which is tentatively assigned to the atypical aquaglyceroporins. Through the combination of physiological, biochemical, genetic, and pathogenicity assays, we demonstrated that pneumococcal AqpC (Pn-AqpC), a representative of the new aquaporin subfamily, facilitates O2 influx into pneumococcal and Pn-AqpC-expressing yeast cells and reconstituted proteoliposomes, thus functioning as an oxygen porin. By transporting O2, Pn-AqpC promoted pneumococcal H2O2 production, resistance to reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as the release of Ply, an important pneumococcal virulence factor (23). Importantly, Pn-AqpC elevated pneumococcal survival in macrophages and increased damage to macrophages, thus contributing significantly to pneumococcal pathogenicity in a murine pneumonia model. Pn-aqpC orthologs are widely distributed in all pneumococcal serotypes and capsule-free strains, implying that this oxygen porin could be a novel virulence-related protein.

RESULTS

Pneumococcus possesses an atypical aquaglyceroporin, Pn-AqpC, representing a novel aquaporin subfamily.

Using the S. oligofermentans aquaporin So-AqpA (I872_01445) as a probe to query the genome of S. pneumoniae D39, an encapsulated serotype 2 strain, three genes (SPD_1320, SPD_1569, and SPD_2011) were hits at amino acid identities of 26%, 95%, and 31%, respectively. Phylogenetically, SPD_1569 clustered with So-AqpA and the water-facilitating aquaporin AqpZ of E. coli and thus was assigned as Pn-AqpA; SPD_2011 was clustered with the glycerol facilitator GlpF of E. coli and assigned as Pn-AqpB. However, SPD_1320 and some glycerol facilitators from other lactic acid bacteria clustered to form a separate branch distantly related to E. coli GlpF (Fig. 1A) and thus assigned as Pn-AqpC. Aquaporins in the Pn-AqpC-affiliated branch were tentatively named atypical aquaglyceroporins and could represent a new aquaporin subfamily. These aquaporins congruously possess YVPR as the substrate-selective residues (Fig. 1B; see also Fig. S1 in the supplemental material), which are distinct from F(H/I)XR in the water-transporting and WG(F/Y)R in glycerol-transporting aquaporins (Fig. 1B and C). Furthermore, the ar/R region diameter size in each Pn-AqpC monomer (Fig. S2A and B) was between those of the E. coli water (Fig. S2C)- and glycerol (Fig. S2D)-transporting aquaporins, implying a different substrate spectrum. Therefore, we investigated the physiological functions of Pn-AqpC, a representative of the new aquaporin subfamily.

FIG 1

Phylogenetic analysis identifies a new subfamily of aquaporins with unique substrate-selective residues. (A) A phylogenetic tree based on the amino acid sequences of the aquaporin orthologs was constructed using the maximum likelihood method with 1,000 replicates. The bar of 0.05 represents evolutionary distance. A dotted line frames the new subfamily of aquaporins (atypical). (B) The amino acid sequences of the aquaporins in panel A were aligned using ClustalW. The E. coli GlpF (b3927) secondary structure (top panel) and the amino acid positions of D39 Pn-AqpC (SPD_1320) (top row of the bottom panel) are shown. Asterisks specify the ar/R region residues, and black lines frame YVPR of the atypical aquaglyceroporins. (C) Distribution of the new subfamily of aquaporins among Lactobacillales. Phylogenetic analysis was implemented as described above for panel A on at most five protein sequences of each genus. Numbers inside parentheses are those containing the YVPR-type aquaglyceroporins; branches within the dark blue pie represent pneumococcal strains.

Structural information on pneumococcal Pn-AqpC. (A) The characteristic Pn-AqpC ar/R region amino acid residues Tyr49, Val223, Pro232, and Arg238, situated at the substrate binding sites, are shown by sticks. (B) The membrane topology was analyzed using TMHMM software. The characteristic ar/R region amino acid residues are shown by blue letters, and the two NPA motifs are shown by orange letters. Download FIG S1, TIF file, 1.1 MB.

Structural homology modeling of pneumococcus Pn-AqpC and channel size comparison with E. coli AqpZ and GlpF. (A) The Pn-AqpC tetramer was constructed by structural homology modeling with SWISS-MODEL by automatically selecting aquaporin-10 (PDB accession no. 6F7H) as the template. (B to D) In each monomer, distances between Y49 and P232 (3.66 Å) and V223 and R238 (9.52 Å) in Pn-AqpC (B), F43 and T183 (5.30 Å) and H174 and R189 (4.20 Å) in E. coli AqpZ (PDB accession no. 1RC2) (C), and W48 and F200 (6.51 Å) and G191 and R206 (11.14 Å) in E. coli GlpF (PDB accession no. 1LDA) (D) were measured using PyMOL. Download FIG S2, TIF file, 2.4 MB.

The absence of Pn-aqpC reduces H2O2 production but promotes the aerobic growth of pneumococcus.

To probe the physiological functions of Pn-AqpC, Pn-aqpC was deleted in S. pneumoniae D39 and its nonencapsulated mutant R6. By reference to S. oligofermentans So-AqpA that facilitates H2O2 permeation, the function of Pn-AqpC in H2O2 transport was first evaluated in R6 and its ΔPn-aqpC mutant carrying a specific cellular H2O2 reporter HyPer gene (24). However, the HyPer reporter detected similar H2O2 influx into ΔPn-aqpC and wild-type (WT) cells when provided exogenous H2O2 (Fig. S3A, bottom), thus excluding a role of Pn-AqpC in H2O2 permeation, whereas significantly lower fluorescence was found in ΔPn-aqpC cells when no exogenous H2O2 was provided (Fig. S3A, top), indicating reduced H2O2 production when Pn-AqpC is absent.

Pn-AqpC is not involved in H2O2 and H2O and glycerol transport. (A) The H2O2 reporter HyPer gene fused to the lactate dehydrogenase gene (ldh) promoter was inserted into pDL278 and then transformed into R6 and the ΔPn-aqpC mutant. The R6 WT-HyPer and ΔPn-aqpC-HyPer strains were statically grown in 10 ml BHI broth in a 100-ml flask. One milliliter of the mid-exponential-phase cells was washed twice and resuspended in 300 μl of PBS. One aliquot was challenged for 30 min with 0.5 mM H2O2 (+H2O2), leaving the other aliquot untreated (−H2O2). After 30 min of air exposure in the dark, the HyPer fluorescence of the cells was examined under a Leica TCS SP8 confocal laser scanning microscope system. Representative fluorescence (left) and corresponding differential interference contrast (DIC) (right) images from three independent experiments are shown. (B to E) Purified Pn-AqpC–10×His proteins were reconstituted into liposomes derived from the E. coli total lipid extract to generate proteoliposomes. To assay water permeability, 100 μl of Pn-AqpC-reconstituted proteoliposomes (B) and Pn-AqpC-devoid liposomes (C) were each rapidly mixed with 100 μl hyperosmolar sucrose to make a final osmotic gradient of 375 mosmol/liter, while to assay glycerol permeability, 100 μl of the glycerol solution was mixed with Pn-AqpC-inserted proteoliposomes (D) and Pn-AqpC-devoid liposomes (E) to make a final osmotic gradient of 375 mosmol/liter. Proteoliposome permeability was measured by monitoring the light scattering intensities using an SX20 stopped-flow spectrometer (Applied Photophysics, Surrey, UK) at 25°C under an emission wavelength of 600 nm. Increased light scattering indicates a vesicle volume decrease that is generated by higher external osmotic pressure-driven water efflux. While water will influx along with a transportable substrate and reswells the shrunken liposome leading to reduction of the light scattering intensity. The kinetics from 5 to 10 measurements were normalized and fitted to an exponential equation, from which the initial rates (k) of substrate permeation were calculated and are shown in the corresponding panels. Experiments were repeated three times, and averages ± standard errors (SE) from one independent assay are shown. Download FIG S3, TIF file, 1.8 MB.

Next, the H2O2 yields of the R6 and D39 wild-type strains and ΔPn-aqpC mutants were assayed in static cultures of 20 and 30 ml of brain heart infusion (BHI) broth in 100-ml flasks, respectively, which build gradient dissolved O2 levels. Surprisingly, the two ΔPn-aqpC mutants both achieved better growth and lower H2O2 yields than the wild-type strains in the two culture volumes (Fig. 2A), while the Pn-aqpC-complemented strains (Pn-aqpC-com) recovered the wild-type phenotype (Fig. 2A). This suggested that Pn-AqpC might facilitate the transmembrane diffusion of O2, a substrate for H2O2 formation. As similarly reduced cellular H2O2 and elevated aerobic growth were determined for the ΔPn-aqpC mutants of the nonencapsulated R6 and encapsulated D39 strains, the Pn-aqpC deletion-caused phenotype changes may not be related to the capsular polysaccharides; thus, strain R6 was investigated for the physiological functions per se of Pn-AqpC in the following experiments, except for animal studies.

FIG 2

Pneumococcal Pn-AqpC acts as an oxygen porin. (A) Growth of R6 (top) and D39 (bottom) wild-type (WT), ΔPn-aqpC, and Pn-aqpC-complemented (com) strains cultured statically in 20 and 30 ml of BHI broth in a 100-ml flask. H2O2 (millimolar) accumulations in the stationary-phase cultures are listed in the table at the bottom, with those of strains R6 and D39 in the top and bottom rows, respectively. (B) An oxygen microsensor Oxy meter was used to measure the residual dissolved O2 in the stationary-phase cultures of the R6 wild type and its derivatives grown in 40 ml BHI broth in a 50-ml centrifuge tube. Oxygen consumption per OD600 of cell mass was calculated by comparison to 283 μM O2 in fresh medium. *, significantly different from other strains. (C) Mid-exponential-phase cultures of the R6 wild type and its derivatives were exposed to air, and the residual O2 in the culture was measured using a phosphorescent oxygen probe. *, significantly different from the wild-type and Pn-aqpC-complemented strains. (D) Protoplasts of S. cerevisiae INVSc1 and INVSc1 carrying the myoglobin gene (INVSc1-myo) or coexpressed with Pn-aqpC (INVSc1-Pn-aqpC-myo) were exposed to air, and the A541 increase per unit of biomass (ΔA541/OD600) was calculated. (E) The residual O2 contents in the culture were determined using a phosphorescent oxygen probe. # and *, significantly different from INVSc1 and INVSc1-myo, respectively. (F and G) The recombinant GST–Pn-AqpC–10×His protein was purified (GST-tag), digested with 100 U thrombin to remove the GST tag to obtain Pn-AqpC–10×His (His-tag) (F), and reconstituted into pneumococcal (Spn) and E. coli liposomes (G). The Pn-AqpC proteins were examined on a 12% SDS-PAGE gel. The protein ladder is shown at the left. Black and gray arrows indicate the macromolecular aggregate and monomer of Pn-AqpC protein, respectively. (H and I) Pn-AqpC facilitating O2 permeation across pneumococcal liposomes (H) and E. coli liposomes (I) was determined using a phosphorescent oxygen probe. *, the fluorescence intensity change at the respective time points was significantly different from that of the Pn-AqpC-devoid liposomes. (J) The R6 wild-type, ΔPn-aqpC, and Pn-aqpC-complemented (Pn-aqpC-com) strains were cultured with shaking at different speeds. The optical density at 600 nm (left) and H2O2 (millimolar) accumulating in stationary-phase cultures (right) were determined. # and *, significantly different from the wild-type and Pn-aqpC-com strains and the respective static cultures, respectively. (K, top and middle) Ten microliters of the mid-exponential-phase cultures in panel J was spotted onto a BHI agar plate and incubated in a 5% O2 environment for 10 h (top), and H2O2 was then determined (middle) as described in Materials and Methods. (Bottom) Chemical H2O2 with known concentrations was used as a reference. All experiments were conducted three times, and averages ± standard deviations (SD) (A, B, D, and J) or averages ± standard errors of the means (SEM) (C, E, H, and I) from one independent assay on triplicate samples are shown. For panels B to D and J, one-way ANOVA and Tukey’s test were performed; for panels E, H, and I, Student’s t test was performed (P < 0.05).

Pneumococcal Pn-AqpC facilitates O2 transport into cells.

To verify the function of Pn-AqpC in transporting O2, a dissolved oxygen microsensor Oxy meter (Unisense, Denmark) was used to measure the residual O2 contents in stationary-phase cultures of pneumococci growing in 40 ml BHI broth in a 50-ml centrifuge tube, and 1.5- to 1.6-fold-lower O2 consumption was determined for the R6 ΔPn-aqpC mutant than for the wild-type and Pn-aqpC-com strains (Fig. 2B). Similarly, using a phosphorescent oxygen probe, a 2.6-fold-lower O2 uptake rate within 3 min was determined for the ΔPn-aqpC mutant than for the wild-type strain, while the Pn-aqpC-com strain recovered O2 uptake levels of the wild type (Fig. 2C). Of note, the H2O2 yields in the wild type (122 ± 3 μM) and the ΔPn-aqpC mutant (18 ± 2 μM) were much lower than the theoretical stoichiometry (258 μM and 173 μM) calculated from the Oxy meter-measured oxygen consumption in the corresponding strains (258 ± 10 μM and 173 ± 21 μM). This indicates that other O2 consumption pathways are present, such as NADH oxidase (Nox) catalyzing the oxidization of NADH to NAD+ and H2O by using O2 as an electron acceptor (25). Therefore, we deleted nox in the wild type and the ΔPn-aqpC mutant, which reduced O2 consumption by 30 ± 3.5 and 34 ± 5.8 μM, respectively. Nevertheless, there should have been other unknown pathways consuming the remaining 106 μM and 121 μM O2 in the wild type and the ΔPn-aqpC mutant, respectively. Moreover, Pn-aqpC deletion did not alter the expression of spxB and lctO, which encode the two major H2O2 production enzymes pyruvate oxidase and lactate oxidase, respectively (Fig. S4). This indicates that the reduced H2O2 production in the ΔPn-aqpC mutant was due to decreased O2 influx instead of reduced expression of the H2O2 production genes.

Effects of Pn-aqpC deletion on the expression of lytA, psaA, pspC, spxB, and lctO in the pneumococcus R6 strain. Total RNAs were extracted from statically grown mid-exponential-phase R6 cells using TRIzol reagent. After quality confirmation on a 1% agarose gel, cDNAs were generated from 2 μg of total RNA with random primers using Moloney murine leukemia virus reverse transcriptase. Quantitative reverse transcription-PCR (RT-qPCR) was implemented using the corresponding primers listed in Table S2 in the supplemental material. Experiments were repeated 3 times on triplicate samples. The transcript copies were calculated per 1,000 16S rRNA copies, and the averages ± SD from three independent experiments are shown. Download FIG S4, TIF file, 0.1 MB.

The O2-facilitating function of Pn-AqpC was further verified by the coexpression of Pn-aqpC and the sperm whale myoglobin (Mb) gene in Saccharomyces cerevisiae INVSc1. Single-Mb-gene-expressing INVSc1-myo and INVSc1 strains were used as controls. In addition, an INVSc1-Pn-aqpC-gfp strain carrying a super folder green fluorescent protein (GFP) gene (sfgfp) fusion to Pn-aqpC was constructed. The expressions of Pn-AqpC and Mb in S. cerevisiae were verified by Western blotting (Fig. S5A), and the cytoplasmic membrane localization of heterologously expressed Pn-AqpC in S. cerevisiae was confirmed via confocal microscopy examination (Fig. S5B). By measuring the characteristic oxymyoglobin (MbO2) absorption at 541 nm in yeast protoplasts (26) (Fig. S5C) and purified MbO2 (Fig. S5D), a significant MbO2 increase (ΔA541/optical density at 600 nm [OD600]) was found in Pn-aqpC-expressing yeast compared to Pn-aqpC-devoid INVSc1 (Fig. 2D). Accordingly, the phosphorescent oxygen probe detected a more rapid decrease of the cultural O2 content of INVSc1-Pn-aqpC-myo than that of INVSc1-myo (Fig. 2E). These results collectively showed that Pn-AqpC facilitates oxygen permeation.

Verification of Pn-aqpC and myoglobin expression in yeast (A and B) and absorption spectra of Pn-aqpC-overexpressing yeast (C) and purified hemoglobin (D). (A) An N-terminally Flag-fused Pn-aqpC gene and a C-terminally 6×His-fused myoglobin gene (myo) from sperm whale (Physeter macrocephalus) were coexpressed in Saccharomyces cerevisiae INVSc1 to obtain strain INVSc1-Pn-aqpC-myo, and the myoglobin gene singly expressed strain INVSc1-myo was used as a background control. After 16 h of culture in SD medium lacking Ura and Leu (SD-Ura-Leu) supplemented with 2% galactose, the two strains were lysed. The same amounts of the cell lysates were used to detect the Pn-AqpC and myoglobin expression by Western blotting using anti-Flag and anti-His antibodies, respectively. (B) Confocal examination shows the cellular localization of Pn-AqpC in S. cerevisiae INVSc1-Pn-aqpC-gfp that carried the green fluorescent protein gene fusion to Pn-AqpC. Representative differential interference contrast (left) and GFP fluorescence (right) images are shown. (C) The same amounts of mid-exponential-phase INVSc1-Pn-aqpC-myo (left) and INVSc1-myo (middle) cells grown in SD-Ura-Leu galactose medium and INVSc1 (right) cells grown in yeast extract-peptone-dextrose (YPD) medium were prepared as protoplasts. The protoplasts were resuspended in isotonic buffer (1.2 M sorbitol, 50 mM magnesium acetate, 10 mM CaCl2), deoxygenated by 7 cycles of vacuum and nitrogen gas flushing, and then fully air exposed with an oxygen pump. The air-exposed cells were dispersed into a 96-well plate, and the absorption spectra were monitored at wavelengths from 300 to 650 nm for 7 min at 60-s intervals using a Synergy H4 hybrid multimode microplate reader (BioTek, Winooski, VT). Experiments were repeated three times, and representative spectra are shown. (D) Hemoglobin (Macklin, Shanghai, China) was dissolved in PBS containing 5% sodium ascorbate to a final concentration of 1 mg/ml, and oxygen was removed by 7 cycles of vacuum-nitrogen flush. The deoxygenated hemoglobin was then air exposed for 15 min using an oxygen pump to obtain oxygenated hemoglobin. The absorption spectra of deoxygenated and oxygenated hemoglobin were monitored under wavelengths of 300 to 700 nm using the Synergy H4 hybrid multimode microplate reader (BioTek). The red dotted line frames the characteristic absorption of oxygenated hemoglobin at 541 nm. Experiments were repeated three times, and a representative spectrum is shown. Download FIG S5, TIF file, 1.4 MB.

Pn-AqpC facilitates oxygen transport across proteoliposomes.

To further confirm that Pn-AqpC facilitates the transport of O2 and other substrates, the recombinant glutathione S-transferase (GST)–Pn-AqpC–10×His protein was purified in the detergent octylglucoside (OG), and the GST tag was then removed (Fig. 2F). Two Pn-AqpC–10×His protein bands of ∼32 kDa and ∼68 kDa were identified as Pn-AqpC by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (Fig. 2F and G; Table S1). Compared to the E. coli AqpZ tetramers that hardly dissociated with 1% SDS due to the strong hydrophobic characteristics (27), the 68-kDa protein was assumed to be a macromolecular aggregate, while the 32-kDa protein was assumed to be a monomer. The purified Pn-AqpC–10×His protein was reconstituted into the membrane lipid of pneumococci and E. coli (Fig. 2G). The phosphorescent oxygen probe was then wrapped within the proteoliposomes and Pn-AqpC-devoid liposomes to detect oxygen contents. This detected 3.2- and 1.5-fold-higher O2 influx in the first 40 s into the pneumococcal and E. coli proteoliposomes than the respective Pn-AqpC-devoid liposomes, respectively (Fig. 2H and I). Notably, 3-fold-higher O2 influx was determined for Pn-AqpC-devoid E. coli than for the pneumococcal liposomes within 80 s (Fig. 2H and I). These results demonstrated that Pn-AqpC sped up O2 permeation across the cellular membrane, and the pneumococcal membrane appears to have lower O2 permeability than that of E. coli.

LC-MS/MS identification of Pn-AqpC–10×His proteins. Download Table S1, DOCX file, 0.02 MB.

Using a stopped-flow apparatus, Pn-AqpC facilitating water and glycerol permeation was measured based on osmosis-driven permeability. However, similar initial rates (k) were determined for proteoliposomes and Pn-AqpC-devoid liposomes in water and glycerol permeation (Fig. S3B to E). Therefore, Pn-AqpC specifically facilitates the permeation of O2 but not water or glycerol.

Pn-AqpC acts as a prominent oxygen facilitator under lower oxygen levels.

Given that O2, particularly at higher concentrations, permeates freely through the cytoplasmic membrane, the range of O2 levels wherein Pn-AqpC plays a role in facilitating O2 was tested by growing the R6 wild-type, ΔPn-aqpC mutant, and Pn-aqpC-com strains under gradient shaking speeds. Although the three strains exhibited similar growth rates under static and 200-rpm shaking conditions, the ΔPn-aqpC mutant grew slightly and markedly better under 60- and 120-rpm shaking conditions (Fig. 2J, left). Accordingly, significantly lower levels of H2O2 were produced in the ΔPn-aqpC mutant cultured under static, 60-rpm, and 120-rpm conditions, but similar H2O2 levels were generated in 200-rpm shaking cultures of wild-type, ΔPn-aqpC mutant, and Pn-aqpC-com strains (Fig. 2J, right). This shows that Pn-AqpC exerts an O2 facilitator role when the bacterium lives under lower O2 levels. Next, the role of Pn-AqpC in the H2O2 production of pneumococci under 5% O2 was tested by mimicking the O2 concentrations in the human lower respiratory tract within the mucus layer or in close contact with pulmonary epithelial cells (11). The same amounts of the wild-type, ΔPn-aqpC mutant, and Pn-aqpC-com cultures were spotted onto a BHI agar plate and incubated in an O2 control in vitro glove box (Coy Laboratory Products). Compared with the wild-type and complemented strains, the ΔPn-aqpC mutant produced almost undetectable H2O2 under 5% O2 (Fig. 2K); therefore, Pn-AqpC could play an important role in pneumococcal infection by facilitating O2 import for H2O2 production, one of the pneumococcal virulence factors.

The substrate-selective residue Pro232 is essential for Pn-AqpC in facilitating O2 permeation.

To determine the key substrate-selective residues for Pn-AqpC in O2 transport, alanine substitution was performed for each YVPR on the shuttle vector pDL278-Pn-aqpC and then transformed into the ΔPn-aqpC mutant to obtain the Tyr49A, Val223A, Pro232A, and Arg238A strains. The four mutants and the Pn-aqpC-com strain were grown in 10 ml BHI broth, and H2O2 yields in the stationary-phase cultures were used as a proxy for O2 uptake. Threefold-reduced H2O2 yields were determined for the Pro232A mutant (0.48 ± 0.39 mM) compared with the Pn-aqpC-com strain (1.46 ± 0.21 mM), whereas no significant change was observed for the Tyr49A (1.38 ± 0.17 mM), Val223A (1.41 ± 0.18 mM), and Arg238A (1.27 ± 0.26 mM) strains.

Furthermore, using the oxygen microsensor Oxy meter, 1.4-fold-lower O2 consumption for the Pro232A mutant was determined than for the wild-type and Pn-aqpC-com strains (Fig. 2B). The phosphorescent oxygen probe also measured 3.3- and 2.3-fold-lower O2 consumption rates within 3 min for the Pro232A mutant than for the wild-type and Pn-aqpC-com strains, respectively (Fig. 2C). These results revealed that Pro232 is the key residue of Pn-AqpC in O2 transport.

Elevated Pn-AqpC protein contents occur in aerobic cultures.

Given the role of Pn-AqpC in O2 transport, its synthesis in response to O2 was determined. Using the photoactivated localization microscopy (PALM) superresolution imaging technique (28), numbers of Pn-AqpC proteins per cell were quantified in anaerobically, statically, and 120-rpm-shaking-grown cultures of the Pn-aqpC-mMaple3 strain, which carried an mMaple3 protein (29) fusion at the C terminus of Pn-aqpC. Figure 3A shows representative PALM images with mMaple3 fluorescence signals; each image included a 2- to 3-cell-constituted cell chain, the typical morphology of pneumococcus. PALM data analysis indicated that the average numbers of Pn-AqpC protein molecules per cell were 23 ± 10 in anaerobic, 56 ± 6 in static, and 94 ± 9 in shaking cultures (Fig. 3A). Catalase treatment did not reduce the Pn-AqpC protein numbers (41 ± 21) in static culture; thus, O2, but not H2O2, seems to induce Pn-AqpC expression.

FIG 3

Oxygen induces Pn-aqpC expression. (A) PALM imaging to assay Pn-AqpC protein expression in the R6 Pn-AqpC-mMaple3 strain grown anaerobically, statically, or with shaking at 120 rpm with the addition of catalase. The mMaple3 fluorescence of mid-exponential-phase cells was observed after 30 min of air exposure in the dark. Pn-AqpC protein numbers were quantified in 18 representative cells, and averages ± SD per cell are shown in parentheses. * and #, significantly different from anaerobically and statically grown cells, respectively (P < 0.05 by one-way ANOVA with Tukey’s test). (B) GFP fluorescence of the mid-exponential-phase cultures was measured in R6 Pn-aqpC-gfp cells cultured anaerobically and shaken with the addition of catalase after air exposure in the dark. The experiments were repeated three times, and averages ± SD from one independent experiment with sextuplicate samples are shown. *, significantly different from the shaking culture cells (P < 0.05 by one-way ANOVA with Tukey’s test). (C, top) Western blot assay of the expression of GFP-tagged Pn-AqpC in the R6 Pn-aqpC-gfp strain grown statically with or without 1 kilounit (KU)/ml catalase and anaerobically with or without 40 μM H2O2 treatment. Band intensities were measured using ImageJ and are shown as percentages of that in static culture. Averages ± SD from three experiments are shown. (Bottom) Total protein separated on an SDS-PAGE gel and used as the protein loading control. (D, top) Statically grown Pn-aqpC-gfp cells were fractionated into cell wall, membrane, and cytoplasmic fractions and subjected to Western blotting using anti-GFP monoclonal antibody. The cell lysate of the wild-type strain (WT) was used as a negative control. (Bottom) SDS-PAGE gel of total protein in each fraction used as the protein loading control. Representative results from three independent experiments are shown in panels A, C, and D.

Oxygen-induced Pn-AqpC expression was further verified by the GFP reporter strain Pn-aqpC-gfp. GFP fluorescence intensities showed a pattern of in anaerobic culture <60 rpm shaking culture <120 rpm shaking culture (Fig. 3B), whereas neither catalase treatment of the static culture nor H2O2 pulsing of the anaerobic culture changed the O2-level-related Pn-AqpC abundances (Fig. 3C). Pn-AqpC was detected exclusively in the cellular membrane fraction (Fig. 3D), confirming its membrane protein identity as predicted by TMHMM (Fig. S1B). These findings indicated that O2 induces the synthesis of Pn-AqpC.

The absence of Pn-AqpC reduces pneumococcal resistance to H2O2 and NO as well as Ply release.

Given that endogenous H2O2 assists pneumococcus in resisting oxidative stress (7), the ΔPn-aqpC mutant reducing H2O2 resistance was presumed to be due to lower H2O2 production. As expected, a lower MIC of H2O2 was determined for the ΔPn-aqpC mutant (5 mM) than for the wild-type strain (8 mM). Consistently, the growth of ΔPn-aqpC in a BHI plate containing 10 mM H2O2 occurred only at a 10−5 dilution compared with the 10−6 dilutions of the wild-type and Pn-aqpC-com strains (Fig. 4A). Moreover, only 2.3% of ΔPn-aqpC mutant cells survived the challenge with 10 mM H2O2, compared with survival rates of >40% for the wild-type and Pn-aqpC-com strains (Fig. 4B). However, 40 μM H2O2 prepulsing significantly increased 10 mM H2O2 survival of the ΔPn-aqpC mutant (Fig. 4B), indicating that Pn-AqpC-promoted endogenous H2O2 production makes pneumococcus withstand exogenous H2O2 challenge.

FIG 4

Absence of Pn-AqpC reduces oxidative resistance and Ply release of pneumococcus. (A) The same amounts of statically grown R6 and its derivatives were 10-fold serially diluted and spotted onto BHI agar containing 10 mM H2O2 or not. Representative growth results from triplicates are shown. (B and C) Strains in panel A were either directly treated or prepulsed with 40 μM H2O2 before being treated with 10 mM H2O2 (B) or 5 mM NO (C). Survival rates are calculated based on CFU. * and #, significantly different from the wild-type and complemented strains and the H2O2 or NO directly treated ΔPn-aqpC mutant, respectively (P < 0.05 by one-way ANOVA with Tukey’s test). (D) Cell autolysis in the stationary and 12-h-post-stationary phases of the strains in panel A was assayed by measurement of the culture OD600. (E) Erythrocytes were incubated with the diluted (fold indicated at the top) 12-h-post-stationary-phase spent cultures of the strains in panel D. Hemolysis was examined after 30 min of incubation at 37°C. (F) Western blot assay of Ply amounts released from the R6 wild type (WT) and the ΔPn-aqpC mutant. 6×His-tagged Ply was expressed in the two strains, which were cultured statically until the stationary and 12-h-post-stationary phases. (Top) The same amounts of 10-kDa ultrafiltration-concentrated cultures were separated on an SDS-PAGE gel, and anti-His antibody was used to detect 6×His-tagged Ply. Band intensities were quantified using ImageJ software, and Ply contents are shown as percentages of the WT Ply-6×His strain (100%) in the stationary-phase spent culture. (Bottom) Total proteins separated on an SDS-PAGE gel were included as a protein loading control. All experiments were executed three times, and averages ± SD from one independent assay on triplicate or quadruplicate samples are shown. Representative results from three independent experiments are shown in panels A, E, and F.

Next, the role of Pn-AqpC in pneumococcal resistance to NO, another oxidant, was assayed as macrophages employ NO-dependent bactericidal mechanisms to clear infecting bacteria (8). Only 5% of the ΔPn-aqpC cells survived 5 mM NO, compared to about 20% survival of the wild-type and Pn-aqpC-com strains (Fig. 4C), suggesting the involvement of Pn-AqpC in NO resistance. Given that the proteins involved in H2O2 resistance also assist E. coli in resisting NO (30), we used 40 μM H2O2 to prepulse the three strains. H2O2 prepulsing increased the NO survival of the ΔPn-aqpC mutant by 6-fold but had no effect on the survival of the wild-type and Pn-aqpC-com strains (Fig. 4C). This shows that lower H2O2 levels induce cross-protection of pneumococci from NO stress.

Stationary-phase cells of pneumococci are usually autolyzed and thus release Ply (31), a major virulence factor. The deletion of Pn-aqpC appeared to significantly alleviate cell autolysis (Fig. 4D) and so may also reduce Ply release and the hemolytic activity of pneumococcus. To test this, 12-h-post-stationary-phase spent cultures of the wild-type, Pn-aqpC deletion, and complemented strains were 2-fold serially diluted, and horse red blood cells were added. Complete erythrocyte lysis was observed in ≤4-fold dilutions of the wild-type and Pn-aqpC-com cultures, but only partial hemolysis occurred in the 2-fold-diluted ΔPn-aqpC culture (Fig. 4E). Consistently, about 2.3-fold less Ply protein was detected in the 12-h-post-stationary-phase spent culture of the ΔPn-aqpC mutant (Fig. 4F). This shows that Pn-aqpC deletion reduces cell lysis as well as Ply release.

Deletion of Pn-aqpC reduces pneumococcal survival in macrophages and damage to macrophages.

Macrophages, the first line of defense of the human immune system, utilize reactive oxygen and nitrogen species to kill invading microbes (8, 9). Given the reduced H2O2 production of the ΔPn-aqpC mutant, the effect of the Pn-aqpC deletion on pneumococcal survival in macrophages was investigated. First, 1 × 105 macrophage RAW 264.7 cells were exposed to the nonencapsulated R6 wild-type, Pn-aqpC deletion, and complemented strains at a multiplicity of infection (MOI) of 100:1. After 1 h of incubation, the bacterial cells attached to and internalized into macrophages were counted, and after additional 1-h and 1.5-h incubations, surviving pneumococcal cells within macrophages were counted. Although the numbers of viable bacterial cells of the three strains all significantly decreased, 4.5- and 3-fold-lower survival rates of the ΔPn-aqpC mutant were determined after additional 1-h and 1.5-h incubations, respectively, than for the wild-type and Pn-aqpC-com strains (Fig. 5A). This validates the contribution of Pn-AqpC to pneumococcal survival in macrophages.

FIG 5

Deletion of Pn-aqpC reduces pneumococcal survival and damage to macrophages and significantly attenuates virulence to mice. (A) Pneumococcal survival in macrophages was assayed by coincubation of 1 × 105 RAW 264.7 cells with the R6 wild type (WT) and derivatives at an MOI of 100:1. After 1 h of incubation, bacterial cells in the culture were removed, those attached to and internalized in macrophages were recorded, and this time point was set as 0 h. After additional 1- and 1.5-h (total, 2- and 2.5-h) incubations in fresh medium containing antibiotics, the pneumococcal cells that survived within macrophages were counted. The nitric oxide (NO) synthetase inhibitor DPI was added to reduce NO production by macrophages. (B to D) Using the same approach as the one described above for panel A, the contribution of Pn-AqpC to R6’s damage to macrophages was evaluated. (B) Live (green)/dead (red) cell staining of macrophages. The addition of 1 KU/ml catalase (cat) was used to assay the role of H2O2. (C) After 16 h of antibiotic treatment, macrophage death (percent) was calculated. (D) Bacterial damage to macrophages was also evaluated by leaked lactate dehydrogenase (LDH) activities. &, significantly different from the respective strains at 0 h (A); *, significantly different from the wild-type and Pn-aqpC-com strains; #, significantly different from that without DPI treatment (A) or the respective strain without catalase addition (C and D) (P < 0.05 by one-way ANOVA and Tukey’s test). (E) BALB/c mice (n = 10; female) were intratracheally infected with 1 × 107 CFU of statically grown D39 wild-type and ΔPn-aqpC strains with or without 40 μM H2O2 pretreatment and the Pn-aqpC-com strain. PBS-administered mice were included as controls. Survival of the infected mice was monitored for up to 12 days, and representative survival curves from two independent experiments are shown. *, significantly different from the wild-type and Pn-aqpC-com strains (P < 0.05 by a log rank Mantel-Cox test). (F) Histopathological observation of the lung tissues of mice that survived PBS administration and ΔPn-aqpC infection and those that died from wild-type and Pn-aqpC-com infections.

To query whether the reduced macrophage survival is caused by the increased NO sensitivity of the ΔPn-aqpC mutant, diphenyleneiodonium chloride (DPI), an inhibitor of inducible nitric oxide synthase (iNOS) (32), was used to inhibit NO production by macrophages. The addition of DPI increased the number of living cells 4.9- and 5.6-fold for the wild-type and Pn-aqpC-com strains, respectively, whereas it enhanced the survival of the ΔPn-aqpC mutant 8.4-fold in macrophages (Fig. 5A), indicating that Pn-AqpC-conferred pneumococcus oxidative stress resistance assists its survival in macrophages.

Given that H2O2 induces macrophage death (33), the role of the deletion of Pn-aqpC in pneumococcal damage to macrophages was examined. Upon bacterial challenging, only 30% of macrophage cells died from coincubation with the ΔPn-aqpC mutant, compared to 72% and 65% cell death from coincubation with the wild-type and Pn-aqpC-com strains, respectively (Fig. 5B and C), whereas the addition of 1 KU/ml catalase reduced macrophage damage from the wild type by 13% (59% with versus 72% without catalase) but did not alleviate the damage from the ΔPn-aqpC mutant (Fig. 5B and C), indicating that Pn-AqpC-promoted H2O2 production has some contributions to macrophage death. The roles of Pn-AqpC and H2O2 in damage to macrophages were also verified by the activities of lactate dehydrogenase in the cultures leaked from macrophages (Fig. 5D). However, the catalase-treated wild-type cells still caused significantly higher macrophage death (59%) than the ΔPn-aqpC mutant (30%), implying that Pn-AqpC itself or other Pn-AqpC-impacted factors, possibly the reduced release of Ply, contribute to macrophages death.

Pn-AqpC is required for pneumococcal virulence in a murine pulmonary infection model.

Given that H2O2 and Ply are major virulence factors (1, 2), and the deletion of Pn-aqpC not only increased the H2O2 and NO susceptibility of but also reduced Ply release by pneumococci, the contributions of Pn-AqpC to pneumococcal virulence were evaluated in a murine pneumonia infection model. BALB/c mice were intratracheally infected with 1.0 × 107 CFU of D39, its Pn-aqpC deletion mutant (pretreated with or without 40 μM H2O2), and the Pn-aqpC-com strain. By monitoring mouse survival for 12 days postinfection, we found that the Pn-aqpC deletion significantly enhanced mouse survival to 78%, compared with 22% and 43% survival rates in the wild-type- and Pn-aqpC-com-infected groups, respectively (Fig. 5E), indicating that Pn-AqpC is involved in pneumococcal pathogenicity. Of note, the survival rate of mice infected with the 40 μM H2O2-pretreated ΔPn-aqpC mutant was reduced to 60%, compared with 78% survival of those infected by the non-H2O2-pretreated ΔPn-aqpC strain, suggesting that low-H2O2-induced oxidative resistance enhances pneumococcal pathogenicity in addition to other virulence factors.

Neither was inflammatory cell immersion (Fig. 5F) observed nor were pneumococci recovered from the lung tissue of the surviving mice infected by the ΔPn-aqpC mutant, whereas 1.35 × 108 ± 0.91 × 108 and 1.54 × 108 ± 0.72 × 108 CFU/ml of pneumococci were recovered from the lungs of dead mice infected by the D39 wild-type and Pn-aqpC-com strains, respectively. These data confirmed the contribution of Pn-AqpC to pneumococcal pathogenicity.

DISCUSSION

To date, 13 and 120 aquaporin isoforms have been identified in humans and plants, respectively, and are delineated into three major subfamilies: the classical water-transporting aquaporins, glycerol-transporting aquaglyceroporins, and AQP supergene channel superaquaporins (14, 15, 34). They facilitate the transmembrane diffusion of water, glycerol, H2O2, CO2, and other small uncharged solutes (14–16, 26, 34, 35). Here, we report a new aquaporin subfamily represented by pneumococcal Pn-AqpC, which functions as an oxygen porin to facilitate oxygen uptake. Phylogenetically, the oxygen porins are distantly related to aquaglyceroporins and possess substrate-selective amino acid residues distinct from those of aquaporins and aquaglyceroporins. Importantly, the oxygen porin Pn-AqpC contributes significantly to the pathogenicity of S. pneumoniae. As depicted in Fig. 6, pneumococcal Pn-AqpC, which is increasingly synthesized under conditions of higher O2 contents, facilitates O2 influx into cells and thus promotes H2O2 production by pneumococcus. Endogenous H2O2 helps pneumococci adapt to higher exogenous H2O2 and NO levels; therefore, the deletion of Pn-aqpC reduced the H2O2 and NO resistance of pneumococci. Accordingly, the presence of Pn-AqpC promotes pneumococcal survival in macrophages and possibly other host immune cells. In addition, the absence of Pn-AqpC alleviates pneumococcal autolysis and, thus, Ply release and significantly reduces pneumococcal damage to macrophages. In support of this, the absence of Pn-AqpC significantly attenuated the virulence of pneumococcus in a murine pneumonia model. Thus, the new subfamily of prokaryotic aquaporins, represented by Pn-AqpC, might be virulence-related proteins.

FIG 6

The newly identified oxygen-facilitating aquaporin Pn-AqpC modulates H2O2 production, ROS and RNS resistance, and pneumolysin (Ply) release and contributes significantly to the pathogenicity of pneumococcus. Pn-AqpC, an atypical aquaglyceroporin, functions as an oxygen porin to facilitate O2 influx and promotes pneumococcus to produce H2O2 via pyruvate oxidase (SpxB) and lactate oxidase (LctO). Endogenous H2O2 endows but deletion of Pn-aqpC reduces pneumococcus resistance to higher exogenous H2O2 and NO levels; therefore, the presence of Pn-AqpC enhances the survival of pneumococci in macrophages. Additionally, the presence of Pn-AqpC promotes pneumococcal cell lysis and, thus, Ply release. As the pneumococcal hemolysin Ply perforates eukaryotic cellular membranes and induces macrophage necroptosis, the presence of Pn-AqpC enhances pneumococcal damage to macrophages. Consistently, the absence of Pn-AqpC significantly attenuates the virulence of S. pneumoniae in a murine pneumonia model.

To our knowledge, Pn-AqpC is the first reported oxygen porin with defined physiological functions. Although O2 freely diffuses across the cytoplasmic membrane (36), assays of both in vivo and heterogeneously expressed yeast and in vitro-reconstituted proteoliposomes all determined that Pn-AqpC increases O2 flux across the cellular membrane (Fig. 2), particularly when pneumococci are grown under lower O2 levels (Fig. 2J and K) similar to those in most host environments (11). In addition, the pneumococcal cellular membrane appears to have lower O2 permeability than that of E. coli, highlighting the role of Pn-AqpC in pneumococci, which could require controllable O2 influx for H2O2 synthesis. Enhanced Pn-AqpC contents were found in aerobically grown pneumococcus (Fig. 3), conforming to its oxygen facilitator mission; however, similar Pn-aqpC transcript levels were found in aerobic and anaerobic cultures (data not shown), implying the posttranscriptional regulation of Pn-AqpC expression. So far, an O2-facilitating function has been reported only for human AQP1 and Nicotiana tabacum PIP1;3 when ectopically expressed in yeast (26). They are affiliated with water-type aquaporins and distantly related to Pn-AqpC at very low protein identities (20% and 14%, respectively) and distinct selective filter residues; therefore, prokaryotic Pn-AqpC represents a novel subfamily of aquaporins. Analogous to other aquaporin orthologs, Pn-AqpC forms a tetramer, as indicated by structural homology modeling implemented in SWISS-MODEL (Fig. S2A) and a macromolecular aggregate formed by the purified Pn-AqpC–10×His protein (Fig. 2F and G). Although O2 is predicted to permeate through the central pore of the four monomers of human AQP1 (36), Pro232 in the Pn-AqpC ar/R region has been determined to be crucial for facilitating O2 transport (Fig. 2B and C). Thus, O2 could be transported through the oxygen porin substrate channel in addition to the tetramer central pore. Thus far, only O2, but neither H2O nor glycerol and H2O2, has been verified as the substrate of Pn-AqpC (Fig. 2; see also Fig. S3 in the supplemental material). Of note, distinct from most other reported aquaporins, Pn-AqpC does not contain cysteine residues and thus is not inactivated by mercury chloride (data not shown).

Significantly, the oxygen porin Pn-AqpC contributes to pneumococcal pathogenicity, as the deletion of Pn-aqpC markedly attenuated lethality to mice (Fig. 5E). Through an exhaustive search, Pn-aqpC orthologs were found in all 8,183 pneumococcal genomes and contigs, which are attributed to 77 capsular serotypes and capsule-free strains. These orthologs exhibit 97% to 100% amino acid sequence identities with Pn-AqpC and 100% identity of YVPR in the ar/R region (Fig. 1C; Data Set S1). Additionally, Pn-AqpC orthologs are widely distributed among members of the genera Streptococcus and Lactococcus of the Streptococcaceae family, the genera Lactobacillus and Pediococcus of the Lactobacillaceae family, the genera Oenococcus and Weissella of the Leuconostocaceae family, and the genus Enterococcus of the Enterococcaceae family (Fig. 1C), so the members of this aquaporin subfamily appear to be restrictively present in facultative anaerobic bacteria, implying that they could contribute to the oxidative adaptation of these bacteria through O2 influx-enabled endogenous H2O2 production. Of note, Pn-AqpC orthologs are particularly prominent in some pathogenic streptococcal species, such as Streptococcus pyogenes and S. mutans (Fig. 1A), implying their association with virulence. Although the non-H2O2-producing species S. mutans also possesses a Pn-AqpC ortholog (SMU_396), its O2-facilitating role is not likely involved in H2O2 production through oxidases, whereas S. mutans encodes H2O-forming NADH oxidase (Nox), which uses O2 to oxidize NADH to NAD+ to achieve cellular redox balance and energy production (37). Deletion of the nox gene reduced O2 consumption by pneumococcus. A previous study also found that O2 promotes O2-tolerant S. mutans growth (38); thus, S. mutans AqpC could also have an important physiological role.

(Sheet 1) Pn-aqpC (Spr1344) of pneumococcus R6 hits for aquaporin orthologs in 8,183 completed genomes and contig sequences of the pneumococcal strains attributed to 77 different pneumococcal serotypes and nondefined serotypes by performing BLASTP analysis. (Sheet 2) Sequences representative of various pneumococcus serotypes possessing Pn-AqpC homologs. (Sheet 3) Amino acid sequences of Pn-AqpC homologs in 8,183 pneumococcus sequenced genomes and contigs. Download Data Set S1, XLSX file, 2.0 MB.

Based on the experimental evidence of pneumococcal survival in and damage to macrophages (Fig. 5A to D), we hypothesize that the mechanistic basis of Pn-AqpC in pneumococcal pathogenicity lies in its control of pneumococcal H2O2 production and Ply release. Compared with other pathogenic bacteria, streptococci are highly capable of resisting oxidative stress via endogenous H2O2-induced resistance to higher levels of exogenous H2O2 (6, 39), and this unique characteristic enables them to defend against the innate immune system of the infected host (9). H2O2 also contributes to pneumococcal virulence by damaging alveolar epithelial cell DNA and suppressing host innate immune systems (4, 5). Consistently, the major H2O2-producing enzyme pyruvate oxidase is crucial for the virulence of S. pneumoniae (40). This work identified that Pn-AqpC, by facilitating O2 uptake, acts as a novel key component in controlling H2O2 production and oxidative stress resistance; thus, the absence of this membrane protein causes S. pneumoniae to be rapidly eliminated by macrophages and reduces damage to macrophages. Survival in macrophages could be important for pneumococcal invasion and is critical for pneumococcal bacteremia and persistence within hosts (10, 41). Remarkably, the presence of Pn-AqpC elevates pneumococcal autolysis and Ply release (Fig. 4E and F), probably due to endogenous H2O2 production, thus increasing the hemolytic activity of pneumococci. Ply, as the major virulence factor of pneumococci, has been known to mediate bacterial transmission, trigger inflammatory responses, and cause macrophage necrosis (3, 42, 43). Rapid autolysis and pneumolysin release were reported to increase the pathogenicity of pneumococcal serotype 1 (44). In addition, no correlations have been found between Pn-AqpC and other identified pneumococcal virulence factors, as the deletion of Pn-aqpC did not alter the transcription of lytA, psaA, pspC, spxB, and lctO (Fig. S4) or the capsule polysaccharide amounts (Fig. S6). Therefore, the virulence relevance of Pn-AqpC lies mainly in its oxygen-transporting function.

Capsular polysaccharide (CPS) amounts were measured with a Stains-all assay. The D39 wild type (WT) and its ΔPn-aqpC and Pn-aqpC-complemented strains (Pn-aqpC-com) were grown on blood agar plates, collected, and resuspended in 150 mM Tris-HCl (pH 7.0)–1 mM MgSO4 to make the cell suspension to an OD600 of 3.5. An aliquot of 1 ml was centrifuged at 13,000 rpm for 10 min, and the pellet was resuspended in 0.5 ml of 150 mM Tris-HCl (pH 7.0)–1 mM MgSO4. The pellet suspension was supplemented with 0.1% (wt/vol) deoxycholate and incubated at 37°C for 15 min to induce cell autolysis; 100 U of mutanolysin, 50 μg of DNase I, and 50 μg of RNase A were then added; and the mixture was incubated for 18 h. The samples were then incubated with 50 μg of proteinase K at 56°C for 4 h. Next, all the samples were 5-fold diluted with 200 μl 150 mM Tris-HCl (pH 7.0), and CPS amounts in each sample were then determined by mixing 250 μl 5-fold-diluted samples with 1 ml of Stains-all solution containing 20 mg of 1-ethyl-2-{3-[1-ethylnaphtho-(1,2-d)thiazolin-2-ylidene]-2-methylpropenyl}naphtho-(1,2-d)thiazolium bromide (Sigma) and 60 μl of glacial acetic acid in 100 ml of 50% formamide and measuring the absorbance at 640 nm. The R6 wild-type strain was included as a background control. All experiments were conducted three times, and the averages ± SD from one independent assay on triplicate samples are shown. Download FIG S6, TIF file, 0.8 MB.

Collectively, Pn-AqpC, by facilitating O2 uptake, modulates H2O2 production and Ply release, the two major virulence factors of pneumococci, and contributes remarkably to pneumococcal virulence. Pn-aqpC orthologs were found in all 8,183 pneumococcal genomes and contigs (Data Set S1). Therefore, the conserved membrane-integrated Pn-AqpC is exposed as a new potential target for fighting against pneumococcal disease.

MATERIALS AND METHODS

Experimental strains and growth.

Experimental strains are listed in Table S2 in the supplemental material. Pneumococcus was grown in brain heart infusion (BHI) broth or agar plates (BD Difco) with 5% sterile defibrinated sheep blood at 37°C with 5% CO2. Pneumococcal strains were grown statically, with shaking, or anaerobically under 100% nitrogen. When required, kanamycin (1 mg/ml) and spectinomycin (300 μg/ml) were added.

Strains, plasmids, and primers used in this study. Download Table S2, DOCX file, 0.03 MB.

Construction of genetically modified strains.

All primers are listed in Table S2. The PCR ligation method (45) was used to construct the Pn-aqpC and nox deletion strains and His-, photoactivatable fluorescent protein mMaple3 (29)-, or super folder green fluorescent protein (sfGFP)-tagged strains of S. pneumoniae. The spectinomycin and kanamycin resistance genes were derived from plasmids pDL278 (46) and pALH124 (47), respectively. The Pn-aqpC gene with its promoter was cloned into pDL278 for complemented strain construction. Alanine substitutions for Tyr49, Val223, Pro232, and Arg238 were implemented on pDL278-Pn-aqpC using a site-directed gene mutagenesis kit (Beyotime, China). Transformation was performed as described previously (48). Correct transformants were confirmed by PCR and DNA sequencing.

Test of the transportable substrates of Pn-AqpC-constituted proteoliposomes.

The purified 10×His-tagged Pn-AqpC protein was reconstituted into liposomes made by the S. pneumoniae cellular membrane lipid (49, 50) and E. coli total lipid extract (Avanti) as previously described (16, 27). Detailed procedures are available in Text S1 in the supplemental material.

Supplemental experimental procedures. Download Text S1, DOCX file, 0.04 MB.

To examine the O2 permeability of Pn-AqpC, cell membrane-impermeable and oxygen-quenchable phosphorescent oxygen probes (Cayman Chemical) were encapsulated into proteoliposomes and Pn-AqpC-devoid liposomes. The proteoliposomes and liposomes were vacuumed and N2 gas flushed for 7 cycles, and 100 μl per well was then dispersed into a 96-well plate (Corning) under air. The fluorescence intensity of the phosphorescent oxygen probe was monitored (excitation at 380 nm and emission at 650 nm) for a recommended delay of 30 μs using a Synergy H4 hybrid multimode microplate reader (BioTek). Water and glycerol permeabilities were assayed using an SX20 stopped-flow spectrometer as previously described (27, 51). The experiments were repeated three times.

PALM imaging.

Mid-exponential-phase Pn-aqpC-mMaple3 cells were exposed to air for 30 min in the dark and then observed using PALM imaging (28) as previously described (12). The superresolution images were constructed using Insight3 software (52), which was kindly provided by Bo Huang (University of California, San Francisco). PALM data analyses such as drift correction, protein abundance, and image rendering were carried out using custom-written Matlab scripts.

Assay of pneumococcal survival in macrophages and damage to macrophages.

Mouse monocyte-macrophage RAW 264.7 cells (1 × 105) were challenged for 1 h with pneumococcus at a multiplicity of infection of 100:1. After removing the bacteria, macrophages were incubated for another 1 and 1.5 h to count CFU of pneumococci within macrophages or for 16 h to determine macrophage death. Detailed procedures are available in Text S1 in the supplemental material.

In vivo mouse infection experiment.

BALB/c mice (specific-pathogen-free [SPF] grade) were purchased from Vital River Company (Beijing, China). Animal experiments were approved by the Biomedical Research Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences. The protocol was approved by the Institutional Animal Care and Use Committee. S. pneumoniae D39 and derivative strains were intratracheally administered to 6- to 8-week-old female BALB/c mice at 1 × 107 CFU in 20 μl phosphate-buffered saline (PBS), and PBS-administered BALB/c mice were included as controls. Mouse survival (10 per group) was monitored for 12 days. Mice were sacrificed under anesthesia, half-lungs were ground for enumerating pneumococcal CFU, and the other halves were used for histopathological observation.

Statistical analysis.

One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test and Student’s t test was performed using PASW Statistics 18 and Excel, respectively. A log rank Mantel-Cox test was performed using GraphPad Prism 8.0. The level of significance was determined at a P value of <0.05.

Other procedures.

Detailed procedures are available in Text S1 in the supplemental material.