ABSTRACT To develop safe and highly effective live vaccines, rational vaccine design is necessary. Here, we sought a simple approach to rationally develop a safe attenuated vaccine against the genome-reduced pathogen Erysipelothrix rhusiopathiae. We examined the mRNA expression of all conserved amino acid biosynthetic genes remaining in the genome after the reductive evolution of E. rhusiopathiae. Reverse transcription-quantitative PCR (qRT-PCR) analysis revealed that half of the 14 genes examined were upregulated during the infection of murine J774A.1 macrophages. Gene deletion was possible only for three proline biosynthesis genes, proB, proA, and proC, the last of which was upregulated 29-fold during infection. Five mutants bearing an in-frame deletion of one (ΔproB, ΔproA, or ΔproC mutant), two (ΔproBA mutant), or three (ΔproBAC mutant) genes exhibited attenuated growth during J774A.1 infection, and the attenuation and vaccine efficacy of these mutants were confirmed in mice and pigs. Thus, for the rational design of live vaccines against genome-reduced bacteria, the selective targeting of genes that escaped chromosomal deletions during evolution may be a simple approach for identifying genes which are specifically upregulated during infection. IMPORTANCE Identification of bacterial genes that are specifically upregulated during infection can lead to the rational construction of live vaccines. For this purpose, genome-based approaches, including DNA microarray analysis and IVET (in vivo expression technology), have been used so far; however, these methods can become laborious and time-consuming. In this study, we used a simple in silico approach and showed that in genome-reduced bacteria, the genes which evolutionarily remained conserved for metabolic adaptations during infection may be the best targets for the deletion and construction of live vaccines.
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