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

## TL;DR

This study compares how two immune genes, CD2AP and BACH2, use genetic code in 49 vertebrates, revealing patterns that may affect protein production in pig macrophages during infection.

## Contribution

The study reveals distinct codon usage preferences for CD2AP and BACH2 across vertebrates, offering a comparative baseline for immune gene research in pigs.

## Key findings

- CD2AP prefers A/T-ending codons, while BACH2 prefers G/C-ending codons across species.
- Both genes show similar levels of codon usage bias despite opposite preferences.
- Compositional background and selective constraints influence codon usage patterns in these immune genes.

## Abstract

Respiratory disease caused by Mycoplasma hyopneumoniae reduces pig welfare and farm productivity. Lung immune cells called alveolar macrophages help control this infection, and their response depends in part on how efficiently key immune genes are made into proteins. The genetic code uses three-letter “words” to build proteins, and different animals can prefer different words even when they produce the same protein building block. In this study, we compared these preferences for two immune-related genes, CD2-associated protein and BTB and CNC homology 2, across 49 vertebrate species. CD2-associated protein tended to use genetic words ending in adenine or thymine, whereas BTB and CNC homology 2 more often used words ending in guanine or cytosine. Although the two genes showed opposite preferences, both displayed a similar overall level of preference across species. These results show that evolution shapes how immune genes are written, which may influence how readily they are produced during infection. The findings provide a comparative reference for pig respiratory immunity research and support gene design for future functional studies.

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.