Role of BgaA as a Pneumococcal Virulence Factor Elucidated by Molecular Evolutionary Analysis

Abstract

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.

Keywords: Streptococcus pneumoniae; bgaA; molecular evolutionary analysis; nanA; pneumococcal cell wall-anchoring proteins.

FIGURE 1

Distribution of genes that encode cell wall-anchoring proteins and percentage of codons that are evolutionarily conserved for these genes. (A) Distribution of genes that encode cell wall-anchoring proteins from pneumococcal strains. The gene locus tag numbers are summarized in Supplementary Table 2. Blue, yellow, and gray represent the presence, pseudogenization, and absence of genes, respectively. (B) Codons of genes encoding cell wall-anchoring proteins evolving under purifying selection were identified using HyPhy software with phylogenetic trees and aligned sequences. Blue, orange, and gray represent the percentage of codons under purifying selection, comparable common codons, and incomparable codons, respectively. The actual numbers and other parameters are listed in Table 1.

FIGURE 2

Bayesian phylogenetic analysis of the bgaA gene. The codon-based Bayesian phylogenetic relationship was calculated using the MrBayes program. Strains with identical sequences are listed on the same branch. S. pneumoniae and S. pseudopneumoniae bgaA genes are shaded in cyan. Other streptococcal bgaA ortholog genes are shaded in magenta. The color gradation of phylogenetic tree represents posterior probability. The scale bar indicates nucleotide substitutions per site.

FIGURE 3

Evolutionarily conserved residues in the BgaA catalytic region. (A) Pictorial representation of the structure of TIGR4 BgaA catalytic region (PDB ID: 4CU6). Glycosyl hydrolase family 2 sugar binding domain, glycosyl hydrolase family 2 domain, glycosyl hydrolase family 2 TIM barrel domain, and DUF4982 are colored in yellow, green, cyan, and magenta, respectively. (B) The side chains of evolutionarily conserved residues are shown as colored stick models. Carbon, nitrogen, and oxygen are shown as green, blue, and red, respectively. (C) BgaA active site is shown as pink stick models, and evolutionary conserved proline residues are in orange. (D) The active site of BgaA. The carbon of the evolutionarily conserved catalytic residues is shown in purple (H484, R602, F603, Y624, E716, T718, H721, and F733). The carbon of the evolutionarily conserved non-catalytic residues is shown in green. Nitrogen and oxygen are shown as blue and red, respectively.

FIGURE 4

Deficiency of bgaA decreases pneumococcal virulence in a mouse model of sepsis. Mice were infected by intravenous injection with 1 × 10⁶ colony forming units (CFU) of S. pneumoniae TIGR4 wild type (n = 21), or ΔbgaA (n = 22), and survival was monitored for 14 days. The difference between infected mouse groups was compared using a log-rank test. The data were obtained from three independent experiments.