Brucella melitensis M5-90Δbp26 as a potential live vaccine that allows for the distinction between natural infection and immunization.

Brucella is Gram-negative intracellular bacterial pathogen that infects humans and animals and contributes to great economic losses in developing countries. Presently, live attenuated Brucella vaccines (Brucella melitensis M5-90) are the most effective means of brucellosis control and prevention in animals. However, these vaccines have several drawbacks, such as an inability to distinguish between a natural infection and immunization and an association with abortions in pregnant animals. Therefore, this study constructed a Brucella M5-90Δbp26 mutant and evaluated its virulence. The survival of the M5-90Δbp26 mutant was attenuated in human placenta trophoblastic 8 cells (HPT-8 cells) and in BALB/c mice, with a high immunoprotectivity noted in mice. Furthermore, safety tests showed that the M5-90Δbp26 mutant was less virulent than the M5-90 vaccine strain. Additionally, an indirect enzyme-linked immunosorbent assay (ELISA) screening was shown to detect the presence of Brucella protein 26 (BP26) with high sensitivity, with M5-90Δbp26 inoculation accompanied with a lack of BP26 expression, and was further confirmed by western blotting. Together, the M5-90Δbp26 mutant and the indirect ELISA can be employed to distinguish vaccinated livestock from infected animals.

Brucella Melitensis 16M Regulates the Effect of AIR Domain on Inflammatory Factors, Autophagy, and Apoptosis in Mouse Macrophage through the ROS Signaling Pathway.

Brucellosis is a highly contagious zoonosis caused by Brucella. Brucella can invade and persist inside host cells, which results in chronic infection. We constructed AIR interference and overexpression lentiviruses to acquire AIR interference, overexpression, and rescue stable expression cell lines. We also established a Brucella melitensis 16M-infected macrophage model, which was treated with either the vehicle control or NAC (ROS scavenger N-acetylcysteine (NAC) for 0, 3, 6, 12, and 24 h. Confocal laser microscopy, transmission electron microscopy, fluorescence quantitative PCR, flow cytometry, ELISA, and Western blot were used to detect inflammation, cell autophagy and apoptosis-related protein expression levels, ROS levels, and the distribution of mitochondria. It was found that after interference and overexpression of AIR, ROS release was significantly changed, and mitochondria became abnormally aggregated. B. melitensis 16M activated the NLRP3/AIM2 inflammatory complex, and induced RAW264.7 cells to secrete IL-1ß and IL-18 through the ROS pathway. B. melitensis 16M also altered autophagy-related gene expression, increased autophagy activity, and induced cell apoptosis through the ROS pathway. The results showed that after B. melitensis 16M infection, ROS induced apoptosis, inflammation, and autophagy while AIR inhibited autophagosome maturation and autophagy initiation. Autophagy negatively regulated the activation of inflammasomes and prevented inflammation from occurring. In addition, mitophagy could promote cell apoptosis.