Analysis of Codon Usage Bias in the Streptococcus pneumoniae Pneumolysin Gene

Streptococcus pneumoniae pneumolysin is a key virulence factor belonging to the cholesterol-dependent cytolysin family, enabling host cell lysis and immune evasion. While synonymous codon usage bias is known to fine-tune virulence gene expression in pathogens, its role in pneumolysin remains uncharacterized. This study presents a comprehensive analysis of codon usage patterns in the pneumolysin gene across 420 curated coding sequences. We found a pronounced preference for A/U-ending codons, significant underrepresentation of CpG dinucleotides, and moderate overall bias (effective number of codons, ENC = 50.28). Neutrality plot, parity rule 2 (PR2) bias, and ENC-plot analyses collectively indicated that natural selection-not mutational pressure-is the dominant evolutionary force shaping this bias. Strikingly, pneumolysin's codon usage showed a significant correlation with the abundant tRNA gene pool of its human host, suggesting an adaptive strategy that may minimize immunostimulation caused by bacterial mRNA release during infection. These findings reveal a balance between translational efficiency, proper protein folding, and immune evasion, thereby providing a functional understanding of pneumolysin evolution and a foundation for practical applications. These include guiding codon-optimized heterologous expression for biochemical studies and enabling codon deoptimization for the design of safer live-attenuated vaccines.