S. pneumoniae strains were cultured as previously described elsewhere (Mori et al., 2012; Yamaguchi et al., 2019b; Yamaguchi et al., 2020). Briefly, the S. pneumoniae strain TIGR4 wild type (WT) and its isogenic bgaA mutant strain [ΔbgaA, (Yamaguchi et al., 2020)] were cultured at 37°C in Todd-Hewitt broth (BD Biosciences, Franklin Lakes, NJ, USA) supplemented with 0.2% yeast extract (THY; BD Biosciences). For the following assays, S. pneumoniae strains were grown to exponential growth phase (OD₆₀₀ = ~0.40) unless otherwise indicated and then resuspended in phosphate buffered saline (PBS) or the appropriate buffer.

Cell culture was performed as previously described (Sumitomo et al., 2012; Yamaguchi et al., 2016). Briefly, human alveolar A549 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS), and human brain endothelial cells (hBMECs) were maintained in RPMI 1640 medium (Fujifilm Wako Pure Chemical Corporation) supplemented with 10% FBS, 10% NuSerum (BD Biosciences), and 1% MEM nonessential amino acids (Merck KGaA, Darmstadt, Germany). hBMECs were seeded and grown in collagen-coated plates. All cells were maintained at 37°C in a 5% CO₂ humidified environment.

Pneumococcal association with A549 cells and hBMECs was performed as previously described, with minor modifications (Yamaguchi et al., 2008; Yamaguchi et al., 2017). Briefly, human epithelial or endothelial cells were seeded at a density of 1 × 10⁵ cells per well in 24-well plates 24 h before infection. In each well, 0.3–6.0 × 10⁷ CFU of S. pneumoniae was added to infect cells. To determine the bacterial association, the infected cells were incubated for 1 h, washed twice with PBS, and then harvested with a solution containing 0.05% trypsin and 0.025% Triton X-100. The associated S. pneumoniae was quantified using serial dilution plating on THY-blood agar.

Bactericidal assays were performed as previously described, with minor modifications (Mori et al., 2012; Yamaguchi et al., 2019a; Yamaguchi et al., 2019b). Briefly, heparinized human blood (190 µL), mouse blood (180 μL), or human neutrophils (2 × 10⁵ cells in 180 μL), and exponential phase bacteria (0.9–2.0 × 10⁴ CFU, 1.5–13.3 × 10⁴ CFU, and 1.8–8.3 × 10⁴ CFU for human blood, mouse blood, and human neutrophils in 10, 20, or 20 μL of PBS, respectively) were mixed in 96-well plates and incubated at 37°C in 5% CO₂ for 1, 2, or 3 h. Viable cell counts were determined by plating diluted samples on THY-blood agar. The growth index was calculated as the number of CFUs at the specified time point divided by the number of CFUs in the initial inoculum.

Human blood was collected via the median cubital vein from healthy donors who agreed to a protocol approved by the Institutional Review Board of Osaka University Graduate School of Dentistry (H26-E43). Human neutrophils were isolated from fresh human blood using Polymorphprep (Alere Technologies AS, Oslo, Norway) according to the manufacturer’s instructions. Mouse blood was collected via cardiac puncture from healthy 6–7 weeks-old CD-1 female mice (Slc:ICR; Japan SLC, Hamamatsu, Japan). All mouse experiments were conducted following a protocol approved by the Animal Care and Use Committee of Osaka University Graduate School of Dentistry (28-002-0).

Mouse infection assays were performed as previously described (Hirose et al., 2018; Yamaguchi et al., 2019a; Yamaguchi et al., 2019b; Yamaguchi et al., 2020). In the sepsis model, female CD-1 mice (Slc:ICR, 6–7-weeks-old) were intravenously infected with 0.4–7.1 ×  10⁶ CFU of S. pneumoniae TIGR4 WT or ΔbgaA strains via the tail vein. To assess bacterial burden, animals were euthanized by lethal intraperitoneal injection of sodium pentobarbital 24  and 36 h after intravenous infection, and blood, brain, lung, liver, spleen, and kidney samples were collected. Bacterial counts in blood or tissue homogenates were determined after plating serial dilutions, with those in the organ corrected for differences in tissue weight. To examine the histopathological features, each tissue specimen was fixed with 4% formaldehyde, embedded in paraffin, and cut into sections that were stained with hematoxylin and eosin solution. The stained tissues were observed using a BZ-X710 microscope (Keyence, Osaka, Japan).

Murine blood was obtained at 12, 24, and 36 h after intravenous infection by cardiac puncture after lethal intraperitoneal injection of sodium pentobarbital. Heparin was added to the blood at a final concentration of 30 U/mL. Total RNA was extracted from leukocytes in the whole blood samples using the PureLink Total RNA Blood Purification Kit and DNase I, Amplification Grade (Thermo Fisher Scientific). RNA integrity was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Full-length cDNA was generated using a SMART-Seq HT Kit (Takara Bio, Shiga, Japan) according to the manufacturer’s instructions. An Illumina library was prepared using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA, USA) according to SMARTer kit instructions. Libraries were sequenced using the Illumina HiSeq 2500 system, with 75 bp single-end reads obtained. The obtained RNA-seq data were analyzed using iDEP ver 0.91, 0.92 or 0.951 (Ge et al., 2018; Ge, 2021). Pathway analysis was performed using the GAGE package and Kyoto Encyclopedia of Genes and Genomes (KEGG) or Reactome datasets on iDEP (Yu and He, 2016; Kanehisa et al., 2017). Raw data generated in this study was submitted to the Gene Expression Omnibus (GEO) dataset under the accession number GSE190418.

Mouse plasma was collected at 24 and 36 h after pneumococcal intravenous infection. Cytokine and chemokine concentrations in mouse plasma were measured by MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel (catalogue number: MCYTOMAG-70K, Merck) and MAGPIX Dx instrument with xPONENT 4.2 (Luminex Japan, Tokyo, Japan) according to the manufacturers’ instructions. Cytokine and chemokine concentrations were inferred from the standards by the acquisition software. We excluded several cytokines and chemokines for which the bead counts were insufficient or several values exceeded the measurement limits from the final data, which precluded accurate statistical analysis. Heatmap was generated using R software ver. 4.1.2 with gplots package.

Mice were euthanized by lethal intraperitoneal injection of sodium pentobarbital 36 h after intravenous infection, and blood aliquots were immediately collected. Blood was drawn from the vena cava of the heart into a syringe containing 3.2% (w/v) sodium citrate. Blood samples were centrifuged for 10 min at 845 × g at room temperature, and the plasma was collected. Prothrombin time and fibrinogen levels were measured using a semi-automated coagulation analyzer KC1 Delta (Tcong Ireland Ltd., Wicklow, Ireland) and the reagents of prothrombin time and fibrinogen (Sysmex, Hyogo, Japan) according to manufacturer’s guidelines (Nakahara et al., 2013).

For platelet aggregation assay, platelet-rich plasma (PRP) was prepared by centrifugation of anticoagulated whole blood at room temperature (104 × g) for 10 min. The blood remaining after removing the PRP was centrifuged at 845 × g for 10 min at room temperature to collect platelet-poor plasma (PPP). To measure platelet aggregation of infected mice, the plasmas were collected at 36 h after intravenous infection. For platelet aggregation assay in vitro, the plasmas were collected from uninfected mice. Obtained PRP was incubated for 30 min at room temperature with S. pneumoniae TIGR4 strains (1.5-2.5 × 10⁶ CFU) WT, ΔbgaA, or ΔbgaA with 40 units of commercial recombinant BgaA (rBgaA; β1-4 Galactosidase S, New England Biolabs Japan Inc., Tokyo, Japan). Platelet aggregation was assessed by light transmission at 37°C using a PRP3000S device (TAIYO Instruments, Inc., Osaka, Japan) according to the manufacturer’s instructions. Platelets were tested for normal responses to collagen (10 μg/mL). The change in light transmission was recorded and compared with that of autologous PPP, which was considered 100% of light transmission.

Statistical analysis of the data obtained from the experiments was performed using the Mann–Whitney U-test or Kruskal–Wallis test with Dunn’s multiple comparisons test. Differences were considered statistically significant at P < 0.05. The tests were carried out using Graph Pad Prism version 7.0d or 8.4.2 (GraphPad Software, Inc., San Diego, CA, USA).

The interaction between S. pneumoniae and the epithelial cells of its host is a prerequisite for pneumococcal disease development. Previous reports have demonstrated that BgaA contributes to the adherence of pneumococcal strains to human epithelial cells. The bgaA mutants in R6 and D39 presented reduced adherence to several epithelial cell lines, such as the human pharyngeal carcinoma cell line Detroit-562 and the human lung carcinoma cell line A549 cells (Limoli et al., 2011; Singh et al., 2014). However, the interaction between pneumococcal BgaA and human endothelial cells has not been previously reported. We confirmed the role of BgaA in pneumococcal association with A549 cells and investigated whether BgaA contributes to pneumococcal association with hBMECs. The ΔbgaA strain showed significantly lower rates of association with A549 and hBMECs compared with the WT strains ( Figure 1A ). These results suggest that BgaA contributes to pneumococcal association with endothelial cells and epithelial cells in vitro.

BgaA inhibits complement-mediated opsonophagocytosis (Dalia et al., 2010). To investigate whether BgaA contributes to pneumococcal survival in blood, we performed bactericidal assays using human blood. After one and two hours of incubation in human blood, there was no significant difference between the survival rates of the WT and ΔbgaA strains. However, after three hours of incubation, the survival rate of ΔbgaA strains was significantly decreased compared with that of WT strains ( Figure 1B ). In addition, we determined the pneumococcal survival rate after incubation for three hours with human neutrophils. The ΔbgaA strain had a significantly lower survival rate than that of the WT after three hours of incubation with human neutrophils, in line with the results of the human blood bactericidal assay ( Figure 1C ). Next, we performed a mouse blood bactericidal assay. In contrast to the effect observed after incubation with human blood, there were no significant differences between the survival of WT and ΔbgaA strains after incubation with mouse blood ( Figure 1D ). Previously, we have shown no large difference in the growth curve of WT and ΔbgaA strains in the THY medium (Yamaguchi et al., 2020). Altogether, these data indicate that BgaA contributes to pneumococcal human-specific resistance to neutrophil killing under a condition without serum components as well as to the evasion from complement-mediated opsonophagocytosis (Dalia et al., 2010).

Our previous study indicated that the virulence of the ΔbgaA strain was significantly lower than that of the WT strain in a mouse sepsis model (Yamaguchi et al., 2020). To elucidate the precise role of BgaA in sepsis, we intravenously infected mice with pneumococcal strains and compared the bacterial burden in the blood, brain, lung, liver, spleen, and kidney 24 and 36 h after infection. In line with the results of previous studies, some of the mice infected with WT S. pneumoniae showed signs of imminent death 36 h after infection (Yamaguchi et al., 2020). At 24 h and 36 h after infection, the amount of CFUs in the blood, lung, liver, spleen, and kidney samples of the WT and ΔbgaA strains were similar ( Figure 2 ). We also examined the histopathological features of the lungs and kidneys, as sepsis often leads to impaired function of these key organs (Fink and Warren, 2014). Histopathological analysis was performed in a blinded fashion by an independent pathologist. Microscopic observation of lungs and kidney samples stained with hematoxylin and eosin revealed that WT-infected mice exhibited more bleeding than PBS-treated or ΔbgaA-infected mice ( Figure 3 ). Moreover, WT-infected mice showed bleeding in the renal bodies in the kidney, and microthrombi in the lungs and kidney, in addition to bleeding. Taken together, these results indicate that the deletion of bgaA reduces tissue damage in infected mice and decreases pneumococcal pathogenicity in a mouse sepsis model.

To investigate the effect of BgaA on the transcriptional response of host cells, leukocyte RNA was obtained from the blood of mice 12, 24, and 36 h after intravenous administration of WT or ΔbgaA pneumococcal strains, or PBS. The transcriptional responses of the murine leukocytes were visualized using a heat map, scatter plots, and principal component analysis ( Figures 4A, B , and Supplementary Figure 1 ). The WT- and ΔbgaA-infected mice showed larger differences at later time points, while all mock mice showed similar tendencies. Venn diagrams showed that WT and ΔbgaA-infected mice shared 22 upregulated and 258 downregulated genes compared with PBS-treated mice at 12 h after infection ( Supplementary Figure 1 ). At 24 h after infection, WT and ΔbgaA-infected mice shared 74 upregulated and 84 downregulated genes compared with PBS-treated mice. Moreover, WT-infected mice presented 15 additional upregulated genes and 53 additional downregulated genes compared with ΔbgaA-infected mice ( Figure 4B and Supplementary Figure 1 ). At 36 h after infection, WT and ΔbgaA-infected mice shared 165 upregulated and 6 downregulated genes compared with PBS-treated mice. WT-infected mice presented upregulated 234 genes and downregulated 382 genes compared with ΔbgaA-infected mice ( Figure 4B and Supplementary Figure 1 ). Pathway analysis of the differentially expressed genes revealed that several signaling pathways associated with host innate immunity were significantly downregulated in WT-infected mice compared with ΔbgaA-infected mice at 24 h and 36 h, whereas there were no significant up- or down-regulated pathways at 12 h post-infection ( Figure 4C ). Expression profiles of TNF-α signaling-related genes at 24 and 36 h after infection were visualized on a KEGG pathway diagram ( Supplementary Figure 2 ). Genes encoding cytokines and chemokines, including TNF-α, CCL-2/MCP-1, and so on, were down-regulated in WT-infected mice compared with ΔbgaA-infected mice, while the expression of TNF-α signaling-related genes was not so largely changed. In addition, Reactome pathway analysis indicated that WT-infected mice presented significantly upregulated platelet activation pathways compared with ΔbgaA-infected mice at 36 h ( Supplementary Table 1 ). Expression profiles of complement and coagulation cascade at 36 h after infection were also visualized on a KEGG pathway diagram ( Figure 5 ). Although the complement and coagulation cascades were not significantly upregulated in WT-infected mice compared with ΔbgaA-infected mice, many genes were upregulated in the coagulation cascade. These results indicate that BgaA inhibits host innate immunity activation and induces coagulation cascades at the RNA level.

Figure 6A shows a heatmap of the data of measured mouse cytokines and chemokines in plasmas obtained using the MAGPIX multiplexing system. The graphs of each cytokine or chemokine are shown in Figure 6B and Supplementary Figure 3 . Although genes encoding TNF-α, IL-1β, IL-15, LIF, CCL-2/MCP-1, CXCL2/MIP-2, M-CSF/CSF-1, and GM-CSF/CSF-2 were down-regulated in RNA-seq analysis ( Supplementary Figure 2 ), no significantly different cytokines and chemokines were observed between WT- and ΔbgaA-infected mice at 24 h and 36 h. These results indicate that BgaA deficiency activates host innate immunity pathways at the RNA level but not the protein level.

Generally, bacterial infections increase the blood concentration of fibrinogen and activate coagulation pathways (Davalos and Akassoglou, 2012). To compare blood coagulation, we measured fibrinogen levels and prothrombin time in mouse blood samples obtained 36 h after infection. WT-infected mice showed higher fibrinogen levels than the ΔbgaA-infected and PBS-treated mice; however, no significant differences were observed in prothrombin time among the groups of mice ( Figure 7A ). Next, we performed a platelet aggregation assay using mouse PRP and PPP 36 h after infection. As shown in Figure 7B , we assessed platelet aggregation rates using pooled PRP and PPP from six mice from each group. WT-infected mice showed reduced platelet aggregation rates compared with those in the other groups, while the level of platelet aggregation of PBS-treated and ΔbgaA-infected mice was comparable. In addition, we assessed platelet aggregation rates using individual PRPs and PPPs from six mice ( Supplementary Figure 4 ). The individual data showed a tendency similar to that of pooled plasmas. To determine whether BgaA affects platelets directly, we performed an in vitro platelet aggregation assay using uninfected mouse PRPs and S. pneumoniae WT, ΔbgaA, and ΔbgaA with rBgaA ( Supplementary Figure 5 ). The assay showed S. pneumoniae strains and rBgaA did not affect platelet aggregation in the PRP. These results indicate a possibility that BgaA does not act directly on platelets but inhibits the aggregation ability via host endothelial cells or other reactions in pneumococcal sepsis.