The protein sequence of SPD_1590 from S. pneumoniae D39 was used as a seed to query the NCBI database using the Protein BLAST tool. Then, multiple sequence alignment and cluster analysis were performed with high-scoring proteins using the software package Clustal-X 2.1.

Streptococcus pneumoniae D39 was cultured in THY medium, composed of Todd-Hewitt broth (Oxoid, United Kingdom) supplemented with 0.5% yeast extract (Oxoid, United Kingdom), or grown on Columbia Agar (Difco, United States) containing 5% sheep blood (Ruite, China) at 37°C in an incubator containing 5% CO₂. Escherichia coli BL21 and DH5α were cultured in Luria-Bertani (LB) medium at 37°C in a shaking incubator. To select positive colonies, erythromycin (Erm; Sigma, United States) at 0.25 μg/ml, chloramphenicol (Cm; Sigma, United States) at 4 μg/ml, or ampicillin (Amp; Sigma, United States) at 100 μg/ml was added to the medium.

The iron-restricted medium was prepared by adding 5% Chelex-100 resin (Bio-Rad, United States) to THY for 8 h with continuous agitation, followed by filter sterilization to remove the resin and supplemented with 100 μM CaCl₂ and 2 mM MgCl₂. When necessary, an iron source like hemin or ferric iron was added to the medium.

To determine bacterial growth curves, the C+Y medium was used (Lacks and Hotchkiss, 1960). This chemically defined medium contained the following ingredients per liter: 5 g casein hydrolysate (vitamin-free casamino acids, Difco, United States), 6 mg tryptophan, 35 mg cystine, 2 g sodium acetate, 8.5 g K₂HPO₄, 0.5 g MgC1₂⋅6H₂O, 2.5 mg CaC1₂, 25 μg MnSO₄⋅4H₂O, 0.5 μg FeSO₄⋅7H₂O, 0.5 μg CuSO₄⋅5H₂O, 0.5 μg ZnSO₄⋅7H₂O, 0.2 μg biotin, 0.2 mg nicotinic acid, 0.2 mg pyridoxine-HC1, 0.2 mg thiamine-HC1, 0.1 mg riboflavin, 0.6 mg calcium pantothenate, 2 g glucose, 0.48 g bovine serum albumin, and 5 g yeast extract. All the bacterial strains and plasmids used were listed in Table 1.

Using flanking homology polymerase chain reaction (LFH-PCR) (Wach, 1996) and the primers listed in Table 2 (1590-P1 to pIB169-spd1590-R), the target gene spd1590 was replaced with an antibiotic resistance cassette gene (erm) to construct Δ1590. The product of LFH-PCR, containing the upstream (621 bp) and downstream (530 bp) sequences of spd1590 and the erm gene (860 bp), was incubated with the WT D39 strain stimulated with CSP₁ (10 ng/ml) at 37°C for 2 h. Then, the transformants were transferred to an Erm-containing Columbia sheep blood agar plate for selection of positive strains, in which spd1590 had been completely replaced by erm. The Δ1590 strain was stocked after seven to eight sequential passages in THY medium with 0.25 μg/ml Erm to stabilize bacterial resistance. For construction of spd1590-overexpressing and -complemented strains, the spd1590 gene was inserted into the pIB169 plasmid, containing an amp resistance cassette, to construct the pIB169-spd1590 plasmid using the ClonExpress II One Step Cloning Kit (Vazyme, China), and the recombinant plasmid was transferred into E. coli DH5α for amplification. The pIB169-spd1590 plasmid was extracted from E. coli DH5α and then transferred to the WT D39 and Δ1590 strains, respectively, which had been stimulated with CSP₁. Transformants were selected on Amp-containing Columbia sheep blood agar plates. All mutant strains were further confirmed by western blotting.

The WT and mutant strains were inoculated into C+Y medium at equal inoculation doses at 37°C in a 5% CO₂ incubator. The optical density at 600 nm (OD₆₀₀) was continuously measured every hour by UV-visible spectroscopy (Evolution 300, Thermo Fisher Scientific, United States) for 12 h. All data were analyzed by GraphPad Prism 6.0 (GraphPad Software, United States).

The target gene spd1590 was cloned from S. pneumoniae D39 genomic DNA by PCR. The PCR product and pBAD/HisA plasmid were digested with restriction enzymes XhoI and KpnI (TaKaRa, Japan), respectively. Next, the fragments were ligated with the ClonExpress II One Step Cloning Kit (Vazyme, China) to construct the fusion plasmid pBAD/HisA-1590. The constructed plasmid was confirmed by DNA sequencing. Then, pBAD/HisA-1590 was transferred into E. coli BL21. BL21/pBAD-HisA-spd1590 was cultured in LB medium with 100 μg/ml Amp at 37°C in a shaking incubator. When the OD₆₀₀ reached 0.6–0.8, 0.2% L-(+)-arabinose was added to the medium to induce the expression of SPD_1590, with continued incubation for 4–6 h. The cells were collected by centrifugation and washed three times with 1× PBS. After resuspending in PBS, the harvested cells were lysed by sonication. The His-SPD_1590 fusion protein was isolated by Ni-NTA, then digested with recombinant Enterokinase (Solarbio, China) to remove the His-tag. The purified protein was verified by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (LTQ Orbitrap XL, Thermo Fisher Scientific, United States) following the method of a previous study (Sun et al., 2011; Li et al., 2013). The purified SPD_1590 was used as an antigen for antibody preparation (Tianjin Sungene Biotech Co., China).

Equivalent quantities of proteins were analyzed by 10–12% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, United States), which were incubated with the anti-SPD_1590 antibody at 4°C. This was followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse as a secondary antibody. The results were visualized using Clarity Western ECL Substrate (Bio-Rad, United States) and captured using ImageMaster 2D Platinum 6.0 (GE Healthcare, United States).

Total proteins were extracted from WT and Δ1590 strains cultivated in THY medium while in the exponential growth phase. A sample of 200 μg total protein was digested with trypsin (Promega, United States) at 37°C for 16 h, then lyophilized. An iTRAQ Reagent 8-plex kit (AB SCIEX, United States) was used to label peptide samples according to the manufacturer’s protocol. All samples were detected using an Orbitrap Fusion Lumos mass spectrometer (Thermo, United States). The parameters used for mass spectrometry have been described previously (Yang et al., 2016). Then, the acquired raw data were identified and quantified via Thermo Proteome Discovery Software 2.1.1.2.1. Quantitative analysis parameters were set as follows: Max. Missed Cleava: 2; Digestion mode: Trypsin; Variable modifications: Oxidation (M), Acetyl (Protein N-term), Phospho (STY); Static modifications: Carbamidomethyl (C), iTRAQ 8-plex.

Gene Ontology (GO) enrichment analysis was performed in Glue GO plug-in of Cytoscape (version 3.5.1) with p < 0.05 to analyze the biological processes of differently expressed proteins (Shannon et al., 2003).

Adhesion and intracellular invasion capabilities were measured according to methods in a previous report (Yu et al., 2017). A549 cells were cultured in DMEM (Thermo Fisher Scientific, United States) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, United States) at 37°C in 5% CO₂. Then, they were gently rinsed twice with PBS. S. pneumoniae strains were suspended in DMEM with 1% FBS and diluted to 1.0 × 10⁷ CFU/ml, while 1 ml dilution was added to each well of A549 cells and incubated for 2 h at 37°C.

For the adhesion assay, A549 cells were washed three times with 1× PBS to remove non-adherent bacteria and digested with 0.25% trypsin (Thermo Fisher Scientific, United States) for 2–3 min. Then, 800 μl cold 1× PBS with 0.025% Triton X-100 (pH 7.4) was added to dissolve A549 cells for 10 min. Next, 100 μl of each cell lysate was diluted with PBS and spread on a Columbia agar plate containing 5% sheep blood.

For the intracellular invasion assay, 15 μg/ml gentamicin (Gen) was added to the medium to kill the bacteria present in each A549 sample, and then the cells were washed with 1× PBS. Next, cells were digested with 0.25% trypsin and dissolved with 0.025% Triton X-100 in PBS. After dissolution, cell lysates were diluted with PBS, and 100 μl of each dilution was spread on a Columbia agar plate containing 5% sheep blood. All plates were cultured at 37°C, and bacterial colonies were counted after 24 h of culture. The adherence and intracellular invasion abilities were measured by determining the number of adherent or invasive bacteria. Each experiment was repeated in triplicate.

For S. pneumoniae, total RNA was extracted with TRIZOL (Invitrogen, United States) after pretreated with 50 mg/ml lysozyme (Sigma, United States). For A549, total RNA was directly extracted with TRIZOL according to the manufacturer’s instructions. The total RNA concentration was measured by NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, United States). The mRNA was reverse-transcribed using a HiScript II Q RT SuperMix for qPCR (+gDNA wiper) Kit (Vazyme, China). The qPCR reaction was performed with the AceQ qPCR kit (Vazyme, China). The expression of target genes was assayed using the StepOne System (Applied Biosystems, United States) and the primers listed in Table 2 (CCL2-F to β-Actin-R or 16S rRNA-F to nanA-R). Actin or 16S rRNA was used as internal controls, and expression levels were analyzed by the comparative critical threshold (2^-ΔΔCT) method (Xie et al., 2011; Livak and Schmittgen, 2012). All experiments were carried out in triplicate.

Bacteria in the exponential growth phase (OD₆₀₀ ∼0.6) were collected by centrifugation and washed three times with 1× PBS that had been pretreated with Chelex-100 resin. After drying with a Scanvac Freeze Dryer (Labgene Scientific, Switzerland), the dry weights were measured. Then, the dry cells were resuspended in 65% nitric acid and heated to 75°C for 20 min. After that, 1.8 ml ddH₂O was added before centrifugation at 13,000 ×g for 5 min. The supernatants were collected and analyzed by ICP-MS (Optima 2000 DV, PerkinElmer, United States). All results were normalized to the dry weight of the cells. Each experiment was repeated in three independent replicates.

The binding of apo-1590 with hemin was detected with a fluorescence spectrometer (F7000, Hitachi, Japan). The parameters were set as follows: the excitation wavelength was 280 nm, the data were captured from 290 to 450 nm at room temperature, and the slit width of excitation was 2.5 nm and that of the emission beam was 10.0 nm. First, 1.2 mM hemin was added dropwise to 1.5 ml 2 μM apo-1590, and the fluorescent spectra were recorded until the fluorescence intensity was stable. Then, the titration curves were analyzed in Origin 8.0, and the binding affinity (Ka) was calculated with a Hill plot.

Proteinase K was diluted with 25 mM Tris–HCl (containing 100 mM NaCl, pH = 7.4) to concentrations of 0, 2.5, 5, 7.5, and 10 μg/ml. Apo-1590 and 1590+hemin were dissolved in 50 mM Tris-HCl (containing 100 mM NaCl, pH = 7.4) to concentrations of 10 μg/ml. Then, 1 μl of diluted proteinase K was added to 9 μl apo-1590 or 1590+hemin to reach final proteinase K concentrations of 0, 0.25, 0.5, 0.75, and 1.0 μg/ml. All samples were heated to 37°C for 1 h. After that, the reaction was terminated by the addition of phenylmethanesulfonyl fluoride (PMSF) or the addition of 5× SDS loading buffer, followed by boiling for 5 min. The results were analyzed with 12% SDS-PAGE.

The secondary structure of apo-1590 and 1590+hemin (6 μM) in 25 mM Tris–HCl (pH = 7.4) was detected with circular dichroism (CD; Applied Photophysics, United Kingdom) spectrometry. The parameters used were derived from a previous study (Li et al., 2014). All captured results were calculated by CDPro and analyzed by Origin 8.0.

Data were statistically analyzed by GraphPad Prism 6.0 (GraphPad Software, United States). Differences between three groups were analyzed by two-tailed, unpaired Student’s t-tests, and data are expressed as mean ± standard (SD). Results were considered significant at p < 0.05.

Amino acid sequence alignment showed that SPD_1590 is highly conserved among Gram-positive bacteria (Figure 1). The region of SPD_1590 from amino acids 16–146 is completely identical to members of the alkaline shock protein (Asp23) family. In Staphylococcus aureus, Asp23 is a membrane-associated protein that is anchored to the membrane via the protein AmaP (Muller et al., 2014). The transcription of asp23 is controlled by the alternative sigma factor σ^B, which is involved in the response of the bacterium to environmental stress. As SPD_1590 has a high similarity to Asp23, we wondered whether SPD_1590 is also a membrane-associated protein.

Subsequently, we found that the homolog of SPD_1590 in S. pneumoniae R6 is SPR_1625, which was annotated as an Asp23/Gls24 family envelope stress response protein. Interestingly, a previous metal-affinity analysis indicated that SPR_1625 contained hemin-binding motifs and showed hemin-binding ability (Romero-Espejel et al., 2013). This initial evidence suggested that SPD_1590 may be involved in hemin transport in Streptococcus as a membrane-associated transporter.

In order to ascertain the biological function of SPD_1590 in vivo, a deletion mutant (Δ1590) was obtained by homologous replacement, and an overexpressing strain (WT+1590) and complemented strain (Δ1590+1590) were constructed by transforming the plasmid pIB169-spd1590 into WT and Δ1590 D39. Western blotting showed that the mutant strains were successfully constructed (Figures 2A,B).

The growth curves of the WT and mutant D39 strains cultured on nutrition-restricted C+Y medium showed that deletion of spd1590 slowed bacterial growth (Figures 2C,D). In contrast, Δ1590+1590 showed a similar growth curve to WT D39, indicating that the complemented expression of 1590 recovered bacterial growth. However, overexpression of SPD_1590 did not increase the growth rate of D39, possibly indicating that SPD_1590 does not alter major metabolic pathways in S. pneumoniae. Notably, when cultured in THY, the growth of Δ1590 is similar to that of WT D39.

To assess whether SPD_1590 is involved in iron uptake, the total metal content of strains WT D39, Δ1590, Δ1590+1590, and WT+1590 was determined by ICP-MS. Compared with the WT strain, Δ1590 had a lower iron content (p = 0.026) and WT+1590 had a higher one (p = 0.044). The complemented expression of SPD_1590 in Δ1590 restored the iron content to a level similar to that in WT D39. In contrast, there was no significant change in Mn content (Figure 2E).

To better understand the role of SPD_1590 in iron uptake, its expression was detected via western blotting in the single mutants ΔpiuA, ΔpiaA, and ΔpitA, and in the triple mutant (ΔpiuA/ΔpiaA/ΔpitA), as these genes encode major iron transporters. SPD_1590 expression in the triple mutant was significantly upregulated, consistent with the previous iTRAQ results of our lab (Yang et al., 2016). In addition, SPD_1590 expression was also increased in the ΔpiuA and ΔpitA strains (Figures 3A,B). PitA is a ferric ion transporter; however, there is hardly any free ferric ion in the host environment. In contrast, hemin and ferrichrome, transported by PiuA and PiaA, respectively, are the major iron sources for bacteria. Therefore, subsequent experiments focused on the relationship between SPD_1590 and PiuA and PiaA.

First, the expression levels of PiuA and PiaA were determined in the WT, Δ1590, and Δ1590+1590 strains. Compared to the WT strain, the expression of PiuA was clearly upregulated in Δ1590, whereas it was similar to that in WT strain and the complemented Δ1590+1590 strain (Figures 3C,D). In contrast, no significant differences were observed in the expression of PiaA in these three strains. Induction of PiuA, a major hemin transporter, in the absence of SPD_1590 suggests that SPD_1590 may also function as a hemin transporter. This potential function was further supported by the fact that, similar to PiuA, SPD_1590 expression was also induced during iron depletion and repressed in the presence of hemin (Figures 3E,F).

To further characterize the interaction between SPD_1590 and hemin, the spd1590 gene was expressed in E. coli BL21 and purified by His-tag affinity chromatography. SDS-PAGE resulted in a single band of approximately 22 kDa, corresponding to the calculated molecular weight of SPD_1590 and indicating the purity of the purified protein (Figure 4A). Western blotting with anti-His and -1590 antibody confirmed the expression of SPD_1590 and the His-tag was successfully removed (Figure 4B).

The binding of the purified SPD_1590 to hemin was measured by fluorescence quenching. The data were curve-fitted to the Hill plot, and the binding constant (Ka) of SPD_1590 to hemin was calculated to be 4.15 × 10⁵ M^-1 (Figure 5A), which is similar to the Ka of PiuA to hemin determined with a similar method (Yao-Jin et al., 2018).

Due to the fact that binding to ligand tightens the binding pocket, the structure of the protein becomes more stable (Stodkilde et al., 2014; Cao et al., 2018). Next, the resistance of apo-SPD_1590 and hemin-bound SPD_1590 to proteinase digestion was measured using various concentrations of proteinase K (Kim et al., 2004). The results indicated that the metal-bound protein was more resistant to proteinase K degradation than apo-SPD_1590 protein under the same experimental conditions (Figure 5B). This effect was more evident at high concentrations of proteinase K. The high resistance ability of hemin-bound SPD_1590 to proteinase K cleavage suggested that hemin binding induced a more compacted protein structure.

To better understand the properties of SPD_1590, its secondary structure was determined by CD spectrometry. Approximately 49.87 ± 1.8% of the apo-protein was folded in α-helices, 27.67 ± 5.4% was in β-sheets, 15.17 ± 4.67% formed β-turns, and 14.4 ± 0.36% was devoid of symmetry. Hemin binding did not result in a significant change in the secondary structure of SPD_1590 (Figure 5C and Table 3). However, changes in the tertiary and quaternary structure of the protein upon hemin binding cannot be ruled out and might explain the differences in the protein properties observed, such as the increased resistance to proteinase degradation.

In addition to being involved in metal ion transport, many ABC transporters of pathogens, including PiuA, are also involved in adhesion to and invasion of host cells (Brown et al., 2001; Durmort and Brown, 2015; Nguyen and Gotz, 2016). Therefore, we investigated whether SPD_1590 was also important in S. pneumoniae infection of human lung carcinoma cells. To this end, the adherence and intracellular invasion of the WT, Δ1590, and WT+1590, as well as ΔpiuA as a positive control, was measured in A549 cells. As shown in Figures 6A,B, spd1590 deletion significantly reduced both the adhesion and intracellular invasion of S. pneumoniae compared to WT. In contrast, spd1590 overexpression resulted in an increase in the number of bacteria adhering to and invading A549 cells. This result indicates that SPD_1590 is important for S. pneumoniae infection of human lung carcinoma cells.

To better understand the role of SPD_1590 during S. pneumoniae infection of human lung carcinoma cells, the expression of inflammatory factors was measured by RT-qPCR in A549 cells infected with the WT, Δ1590, and WT+1590 strains. As shown in Figures 6C,D, compared with the WT strain, the expression of IL-1β, CCL2, and IL-6 was significantly reduced in cells infected with Δ1590 and elevated in cells infected with WT+1590. This indicates that SPD_1590 alters the inflammatory response of A549 cells to S. pneumoniae.

Finally, we explored the role of SPD_1590 in the context of the bacterial metabolism by using iTRAQ-based proteomics to identify differentially expressed proteins between the WT and Δ1590 strains. Using isotopic labels and three biological replicates, 1259 and 1275 proteins were detected in the WT and Δ1590 strains, respectively. Relative quantitative analysis revealed 86 differentially expressed proteins (p < 0.05) in Δ1590 compared with the WT, including 24 upregulated proteins (≥1.20-fold) and 62 downregulated proteins (≤0.83-fold). Detailed information on the differentially expressed proteins is provided in Supplementary Table S1.

The biological processes enriched in the 86 differentially expressed proteins were determined by GO analysis and included carbohydrate metabolism, transcription regulation, phosphate-containing compound metabolism, oxidation-reduction reactions, and nucleic acid phosphodiester bond hydrolysis (Table 4). Many proteins involved in carbohydrate metabolism, [Beta-galactosidase (putative), Beta-galactosidase 3, and Galactose-6-phosphate isomerase subunit LacB] and carbohydrate transport [five components of phosphotransferase (PTS) system] were downregulated, suggesting that Δ1590 may have a low metabolic rate. This result is consistent with the fact that Δ1590 showed a reduced growth rate compared to that of WT D39 in C+Y medium. In addition, four kinases including three histidine kinases regulating many important biological pathways and bacterial pathogenesis, were altered in Δ1590. Meanwhile, six proteins involved in oxidation-reduction process were changed indicating that cellular reactive oxygen species (ROS) altered by the deletion of spd1590. Additional proteins were notable even though they were not part of specific biological pathways. For instance, the expression levels of a cell wall surface anchor family protein (SPD_0335) and of the pneumococcal surface protein A (PspA) were 0.56- and 0.57-fold lower, respectively, in the mutant strain. These two proteins contribute to S. pneumoniae virulence and pathogenicity, suggesting that their reduced expression may be related to the attenuation of virulence in Δ1590.

To further explore this, the expression levels of several virulence factors were determined by RT-qPCR in D39 and Δ1590. Compared to levels in WT D39, levels of the virulence factors pspA, nanA, lytA, and cbpD were significantly reduced at mRNA level in Δ1590 (Figure 6E). In addition, the decrease in pspA was consistent with the findings of the above mentioned proteomics analysis. As these virulence factors contribute to adherence, colonization, and migration, this result may reflect the mechanism by which spd1590 expression influences S. pneumoniae pathogenicity in A549 cells and the cell inflammatory response.