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.