tRNA modifications enable codon usage signatures to coordinate bacterial virulence in Salmonella

Pathogens must express virulence programs while competing for limited resources and adapting to nutrient shifts. Many virulence loci exhibit atypical codon usage, but the functional consequences of this bias remain unclear. Here, we mapped unexpected rich landscapes of diverse, functionally distinct codon usage patterns across the genomes of diverse bacterial species. In Salmonella, virulence genes formed a unique signature enriched for rare tRNAs and favoring wobble decoding. This alleviated competition with highly expressed genes, at the cost of increased dependence on specific tRNA modifications for efficient and accurate decoding. In a Salmonella systemic mouse infection model, loss of i6A37 and mnm5s2U34 modifications (miaA and mnmEG mutants) abolished virulence and selectively suppressed virulence proteins. Crucially, using recoded fluorescent proteins, we showed that virulence codon bias, by avoiding codons with high turnover, sustained increased translation rates during amino-acid starvation. Together, these findings indicate that virulence codon bias is conserved across pathogens, ensures robust expression during starvation and couples tRNA modifications to pathogenic functions, highlighting tRNA modification pathways as potential targets for broad anti-virulence strategies.