Comparative Codon Usage Bias of CD2AP and BACH2 Across 49 Vertebrates: Implications for Porcine Macrophage Immunity in Mycoplasma hyopneumoniae Infection

Alveolar macrophages orchestrate phagocytosis and inflammatory programs during respiratory infection. CD2-associated protein (CD2AP) and BTB and CNC homology 2 (BACH2) are immune-related genes involved in cytoskeletal organization/vesicular trafficking and transcriptional regulation, respectively, but the coding-level constraints shaping their synonymous-site architecture remain unclear. Here, we profiled codon usage bias (CUB) of CD2AP and BACH2 across 49 vertebrate species using nucleotide composition, relative synonymous codon usage, and complementary codon bias diagnostics. Across species, BACH2 preferentially used G/C-ending codons with higher GC3s, whereas CD2AP was enriched for A/T-ending codons with lower GC3s. Both genes showed weak-to-moderate CUB (high ENC and modest CAI). ENC–GC3s and PR2 analyses indicated a strong compositional background at third codon positions, while neutrality analysis yielded shallow GC12–GC3 slopes, suggesting overall coding constraints, with compositional effects acting as a background influence and selective constraints possibly contributing to GC1/GC2 patterns. CD2AP deviated more from composition-only expectations than BACH2, suggesting greater gene-specific modulation. Phylogenetic reconstruction placed Sus scrofa within mammalian clades for both genes. In conclusion, CD2AP and BACH2 display opposite third-base codon-ending preferences across vertebrates, with CD2AP favoring A/T-ending codons and BACH2 favoring G/C-ending codons. This provides a comparative baseline for codon usage analyses of macrophage-relevant immune genes.