Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral streptococcus species, mitis group. This pathogen is a leading cause of community-acquired pneumonia, which often evades host immunity and causes systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a β-helical cell surface protein contributing to pneumococcal adhesion to and invasion of human epithelial cells in addition to its survival in blood. In the present study, we investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis indicated that the pfbA gene is highly conserved in S. pneumoniae and Streptococcus pseudopneumoniae within the mitis group. Our in vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to pneumococcal survival. We found that PfbA activates NF-κB through TLR2, but not TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the survival of the S. pneumoniae Δ pfbA strain as compared to a control peptide treatment, whereas the treatment did not affect survival of a wild-type strain. In a mouse pneumonia model, the host mortality and level of TNF-α in bronchoalveolar lavage fluid were comparable between wild-type and Δ pfbA -infected mice, while deletion of pfbA decreased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis model, the Δ pfbA strain demonstrated significantly increased host mortality and TNF-α levels in plasma, but showed reduced bacterial burden in lung and liver. These results indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting host cell phagocytosis, excess inflammation, and mortality by interacting with TLR2.

Streptococcus pneumoniae is a major cause of pneumonia, sepsis, and meningitis. Previously, we identified a novel virulence factor by investigating evolutionary selective pressure exerted on pneumococcal choline-binding cell surface proteins. Herein, we focus on another pneumococcal cell surface protein. Cell wall-anchoring proteins containing the LPXTG motif are conserved in Gram-positive bacteria. Our evolutionary analysis showed that among the examined genes, nanA and bgaA had high proportions of codon that were under significant negative selection. Both nanA and bgaA encode a multi-functional glycosidase that aids nutrient acquisition in a glucose-poor environment, pneumococcal adherence to host cells, and evasion from host immunity. However, several studies have shown that the role of BgaA is limited in a mouse pneumonia model, and it remains unclear if BgaA affects pneumococcal pathogenesis in a mouse sepsis model. To evaluate the distribution and pathogenicity of bgaA, we performed phylogenetic analysis and intravenous infection assay. In both Bayesian and maximum likelihood phylogenetic trees, the genetic distances between pneumococcal bgaA was small, and the cluster of pneumococcal bgaA did not contain other bacterial orthologs except for a Streptococcus gwangjuense gene. Evolutionary analysis and BgaA structure indicated BgaA active site was not allowed to change. The mouse infection assay showed that the deletion of bgaA significantly reduced host mortality. These results indicated that both nanA and bgaA encode evolutionally conserved pneumococcal virulence factors and that molecular evolutionary analysis could be a useful alternative strategy for identification of virulence factors.

Streptococcus pneumoniae is a major cause of invasive diseases such as pneumonia, meningitis, and sepsis, with high associated mortality. Our previous molecular evolutionary analysis revealed that the S. pneumoniae gene bgaA, encoding the enzyme β-galactosidase (BgaA), had a high proportion of codons under negative selection among the examined pneumococcal genes and that deletion of bgaA significantly reduced host mortality in a mouse intravenous infection assay. BgaA is a multifunctional protein that plays a role in cleaving terminal galactose in N-linked glycans, resistance to human neutrophil-mediated opsonophagocytic killing, and bacterial adherence to human epithelial cells. In this study, we performed in vitro and in vivo assays to evaluate the precise role of bgaA as a virulence factor in sepsis. Our in vitro assays showed that the deletion of bgaA significantly reduced the bacterial association with human lung epithelial and vascular endothelial cells. The deletion of bgaA also reduced pneumococcal survival in human blood by promoting neutrophil-mediated killing, but did not affect pneumococcal survival in mouse blood. In a mouse sepsis model, mice infected with an S. pneumoniae bgaA-deleted mutant strain exhibited upregulated host innate immunity pathways, suppressed tissue damage, and blood coagulation compared with mice infected with the wild-type strain. These results suggest that BgaA functions as a multifunctional virulence factor whereby it induces host tissue damage and blood coagulation. Taken together, our results suggest that BgaA could be an attractive target for drug design and vaccine development to control pneumococcal infection.

20Streptococcus pneumoniae is a Gram-positive bacterium belonging to the oral 21 streptococcus species, mitis group. This pathogen is a leading cause of 22 community-acquired pneumonia, which often evades host immunity and causes 23 systemic diseases, such as sepsis and meningitis. Previously, we reported that PfbA is a 24 β-helical cell surface protein contributing to pneumococcal adhesion to and invasion of 25 human epithelial cells in addition to its survival in blood. In the present study, we 26 investigated the role of PfbA in pneumococcal pathogenesis. Phylogenetic analysis 27 indicated that the pfbA gene is specific to S. pneumoniae within the mitis group. Our in 28 vitro assays showed that PfbA inhibits neutrophil phagocytosis, leading to 29 pneumococcal survival. We found that PfbA activates NF-κB through TLR2, but not 30 TLR4. In addition, TLR2/4 inhibitor peptide treatment of neutrophils enhanced the 31 survival of the S. pneumoniae ∆pfbA strain as compared to a control peptide treatment, 32whereas the treatment did not affect survival of a wild-type strain. In a mouse 33 pneumonia model, the host mortality and level of TNF-α in bronchoalveolar lavage 34 fluid were comparable between wild-type and ∆pfbA-infected mice, while deletion of 35 4 pfbA increased the bacterial burden in bronchoalveolar lavage fluid. In a mouse sepsis 36 model, the ∆pfbA strain demonstrated significantly increased host mortality and TNF-α 37 levels in plasma, but showed reduced bacterial burden in lung and liver. These results 38 indicate that PfbA may contribute to the success of S. pneumoniae species by inhibiting 39 host cell phagocytosis, excess inflammation, and mortality. 40 41 Importance 42Streptococcus pneumoniae is often isolated from the nasopharynx of healthy 43 children, but the bacterium is also a leading cause of pneumonia, meningitis, and sepsis. 44In this study, we focused on the role of a cell wall anchoring protein, PfbA, in the 45 pathogenesis of S. pneumoniae-related disease. We found that PfbA is a 46 pneumococcus-specific anti-phagocytic factor that functions as a TLR2 ligand, 47indicating that PfbA may represent a pneumococcal-specific therapeutic target. 48However, a mouse pneumonia model revealed that PfbA deficiency reduced the 49 bacterial burden, but did not decrease host mortality. Furthermore, in a mouse sepsis 50 model, PfbA deficiency increased host mortality. These results suggest that S. 51 5 pneumoniae optimizes reproduction by regulating host mortality through PfbA; 52 therefore, PfbA inhibition would not be an effective strategy for combatting 53 pneumococcal infection. Our findings underscore the challenges involved in drug 54 development for a bacterium harboring both commensal and pathogenic states. 55 56