A novel Brucella T4SS effector RS15060 acts on bacterial morphology, lipopolysaccharide core synthesis and host proinflammatory responses, which is beneficial for Brucella melitensis virulence

Brucella relies on the type IV secretion system (T4SS) to establish replication niches within host cells. However, the Brucella T4SS effectors and their functions have not been fully identified. In this study, we investigated the function of Brucella RS15060, a novel T4SS effector discovered in our previous study, on the bacterial biological characteristics and pathogenesis by construction of the gene deletion and complementation strains. We found that deletion of the rs15060 gene weakened abilities of Brucella to replicate within host cells and establish chronic infection in mice but enhanced abilities to adhere/invade HeLa cells and evade lysosomal degradation in the early stage of infection. In addition, the rs15060 deletion Brucella strain showed significant changes in bacterial shape, cell wall thickness, and sensitivity to bactericidal factors. Furthermore, the rs15060 deletion strain showed an increased synthesis of bacterial lipopolysaccharide core and induced a stronger host's inflammatory response. The Brucella rs15060 complementation strain restored the altered biological characteristics. Moreover, BLASTP prediction and 3D structure simulation revealed that the Brucella RS15060 contains NAD (P)-binding and active motifs in structure, which are important for proteins to exert NAD dependent epimerase/ dehydratase activity. The complementation strain with mutation on NAD(P)-binding and/or active motifs of RS15060 did not restore the altered characteristics, suggesting that the Brucella RS15060 is a potential NAD dependent epimerase/dehydratase, and the predicted NAD(P)-binding and/or active motifs play an important role on bacterial cell wall and LPS core synthesis, which is crucial for maintaining bacterial morphology and exerting virulence.