A novel aquaporin Aagp contributes to Streptococcus suis H₂O₂ efflux and virulence

Figures & data

Figure 1. The transcriptional landscape of S. suis oxidative response. (a) RNA-Seq analysis was performed to explore the global H₂O₂ stress response of S. suis. The upregulated genes are shown in green, and the downregulated genes are shown in red. The detailed information and fold change of genes shown in this figure are summarized in table 1. (b) The survival rate of WT, ΔAagp, Δexo III, ΔAguB, and ΔmetQ was determined after 25 min treated with 25 mM H₂O₂. The statistical analyses were performed with a two-tailed unpaired t test. “****” indicate p < 0.001.

Table 1. The key differentially expressed genes under H₂O₂ stress^a.

ID	Fold Change (H2O2/THB)	Product	Function
BFP66_RS01250	3.66	aquaporin Aagp;	H2O2 efflux
BFP66_RS01545	−3.62	transcriptional regulator PerR;	oxidative stress regulation
BFP66_RS09925	−1.93	transcriptional regulator mntR;	oxidative stress regulation
BFP66_RS08225	1.35	methionine ABC transporter, MetQ	methionine transporter, converting H2O2 to H2O
BFP66_RS06520	2.01	N-carbamoylputrescine amidase;	converting agmatine to putrescine, which protects DNA from damage
BFP66_RS05940	2.89	exodeoxyribonuclease III;	DNA repair
BFP66_RS06160	2.19	N-acetyl-beta-hexosaminidase;	producing sugar by cell wall hydrolyzation
BFP66_RS04160	2.61	L-lactate dehydrogenase LDH;	generating NAD^+/FAD^+/NADP^+ for biosynthesis of amino acids and nucleic acids
BFP66_RS01580	2.02	NADP-dependent oxidoreductase;
BFP66_RS05715	2.30	NADH oxidase;
BFP66_RS07745	2.09	nicotinate phosphoribosyltransferase;
BFP66_RS06935	2.21	flavodoxin;
BFP66_RS01665	3.07	fructose-bisphosphate aldolase;	glycolysis
BFP66_RS07495	2.61	2,3-bisphosphoglycerate-dependent phosphoglycerate mutase;
BFP66_RS06470	2.19	pyruvate kinase;
BFP66_RS06865	2.36	enolase;
BFP66_RS02590	2.86	triose-phosphate isomerase;
BFP66_RS00790	2.16	type I glyceraldehyde-3-phosphatedehydrogenase;
BFP66_RS00845	−2.35	sugar ABC transporter substrate-binding protein;	sugar ABC transporters
BFP66_RS00850	−2.22	sugar ABC transporter permease;
BFP66_RS02995	−8.65	sugar ABC transporter permease;
BFP66_RS07040	−15.41	sugar ABC transporter permease;
BFP66_RS07045	−10.83	sugar ABC transporter substrate-binding protein;
BFP66_RS08960	−2.20	sugar ABC transporter substrate-binding protein;
BFP66_RS00910	−7.00	PTS sugar transporter subunit IIC;	PTS system
BFP66_RS02210	−3.46	PTS system mannose/fructose/N-acetylgalactosamine-transporter subunit IIB;
BFP66_RS02215	−1.90	PTS mannose/fructose/sorbose/N-acetylgalactosamine transporter subunit IIC;
BFP66_RS02695	−6.00	PTS glucose transporter subunit IIBC;
BFP66_RS03575	−14.07	PTS N-acetylgalactosamine transporter subunit IIA;
BFP66_RS04315	−3.47	PTS lactose/cellobiose transporter subunit IIA;
BFP66_RS04320	−3.65	PTS lactose transporter subunits IICB;
BFP66_RS08510	−10.66	PTS cellobiose transporter subunit IIC;
BFP66_RS08530	−53.00	PTS cellbiose transporter subunit IIC;
BFP66_RS08570	−5.96	PTS beta-glucoside transporter subunit EIIBCA;
BFP66_RS09840	−2.24	PTS sugar transporter subunit IIC;
BFP66_RS09850	−4.61	PTS lactose/cellobiose transporter subunit IIA;
BFP66_RS09855	−3.09	PTS sugar transporter subunit IIB;
BFP66_RS05750	−3.95	aquaporin family protein GlpF;	glycerol metabolism
BFP66_RS05760	−1.66	glycerol kinase GlpK;
BFP66_RS05755	−3.12	type 1 glycerol-3-phosphate oxidase GlpO;
BFP66_RS05785	−12.96	glycerol dehydrogenase GldA;
BFP66_RS05790	−51.82	acetaldehyde/alcohol dehydrogenase;
BFP66_RS05800	−2.54	Formate C-acetyltransferase/glycerol dehydratase family glycyl radical enzyme;
BFP66_RS09160	−2.28	dihydroxyacetone kinase transcriptional activator DhaS;

Note: ^a The threshold of p < 0.01 was used to identify the differentially expressed genes.

Figure 2. S. suis Aagp belongs to atypical aquaglyceroporins. The S. suis Aagp transmembrane helices were predicted (top panel). Asterisks specify the ar/R region residues, and black boxes mean the atypical aquaglyceroporins’ substrate-selective residues (YVPR).

Figure 3. Aagp, a novel aquaporin, is widely distributed in S. suis. (a) The distribution of Aagp was assessed in 126 S. suis strains by examining Aagp sequences obtained from the NCBI database. (b) The survival rates of P1/7 and ΔAagp_P1/7 were determined after 25 min treated with 25 mM H₂O₂. The statistical analyses were performed with a two-tailed unpaired t test. “**” indicate p < 0.01.

Figure 4. Aagp facilitates H₂O₂ efflux. (a) The survival rates of WT and ΔAagp were determined after 25 min treated with 25 mM H₂O₂. (b) The H₂O₂ concentrations of WT and ΔAagp were measured after 25 min treated with 25 mM H₂O₂. (c) The survival rates of WT and Aagp^mut were determined after 25 min treated with 25 mM H₂O₂. (d) The H₂O₂ concentrations of WT and Aagp^mut were measured after 25 min treated with 25 mM H₂O₂. (e) The response to H₂O₂ between the wild type and Aagp mutations via the Oxford cup test. The statistical analyses were performed with a two-tailed unpaired t test. “*” indicates p < 0.05 and “**” indicates p < 0.01.

Figure 5. Aagp is involved in glycerol transport. The growth curves of WT (a) and ΔAagp (b) cultured in 1/3 THB, 1/3 THB containing 10 mM glycerol or 100 mM glycerol were shown. (c) S. suis strain GZ0565 possesses a phosphorylation pathway for glycerol metabolism. The downregulated genes are shown in red in H₂O₂ treatment compared with THB condition by RNA-Seq analysis. (d) The H₂O₂ concentrations of WT and ΔAagp cultured in 1/3 THB, 1/3 THB containing 10 mM glycerol or 100 mM glycerol were determined. The statistical analyses were performed with a two-tailed unpaired t test. “*” indicates p < 0.05.

Figure 6. Aagp contributes to S. suis virulence in a mouse infection model. Five mice per group were injected intraperitoneally with 1.5 × 10⁸ CFU of WT and ΔAagp. All mice were euthanized at 24 h post-infection. Bacteria from blood, liver, spleen, kidney and brains were plated onto THA, and colonies were expressed as Log₁₀ CFU/mg or Log₁₀ CFU/mL. The statistical analyses were performed with a two-tailed unpaired t test. “***” indicate p < 0.001.

Data Availability statement

The data supporting this study’s findings are available from the corresponding author upon reasonable request.