Identification of novel genes essential for Brucella abortus to establish infection by signature-tagged mutagenesis.

Brucella is a facultative intracellular bacterium, causing brucellosis, an important zoonosis worldwide. Brucella has no classic virulence factors, thus virulence is dependent on invasion of host cells and subsequent intracellular replication. Identification of key genes involved in Brucella virulence is important to further elucidate its pathogenesis. In this study, signature-tagged mutagenesis was used to identify novel genes involved in B. abortus infection in a mouse model. In total 3600 mutants were obtained, of which 56 were identified as attenuated mutants. Furthermore, 53 genes were identified to be inactivated by transposon insertion, including 19 genes previously reported to be essential for Brucella virulence and 34 others that were newly identified in this study. These genes were catalogued into 16 functional classifications, except for three that were not cited in the Clusters of Orthologous Groups database. Bioinformatics analysis revealed that energy production and conversion, amino acid transport and metabolism, as well as inorganic ion transport and metabolism were predominant functional classifications, suggesting that genes involved in these functions were crucial for Brucella virulence. In addition, the function of the identified pyruvate carboxylase (pyc) gene in bacterial virulence was confirmed using an allelic replacement pyc mutant and a mouse model. These findings provide novel genetic information associated with Brucella infection.