Brucella transfers effectors into host cells, manipulating cellular processes to its advantage; however, the mechanism by which effectors regulate cellular processes during infection is poorly understood. A growing number of studies have shown that apoptosis and autophagy are critical mechanisms for target cells to cope with pathogens and maintain cellular homeostasis. BtpB is a Brucella type IV secretion system effector with a complex mechanism for manipulating host infection. Here, we show that the ectopic expression of BtpB promoted DNA fragmentation. In contrast, an isogenic mutant strain, ΔbtpB, inhibited apoptosis compared to the wild-type strain B. suis S2 in RAW264.7 cells. In addition, BtpB inhibited autophagy, as determined by LC3-II protein levels, the number of LC3 puncta, and p62 degradation. We also found that BtpB reduced autophagolysosome formation and blocked the complete autophagic flux. Moreover, our results revealed that the autophagy inhibitor, chloroquine, reduces Brucella’s intracellular survival. Overall, our data unveil new mechanisms of virulence implicating the effector BtpB in regulating host intracellular infection.

The arsenic acid-resistant (ArsR) family transcriptional regulators are widely distributed in microorganisms, including in the facultative intracellular pathogen Brucella spp. ArsR proteins are implicated in numerous biological processes. However, the specific roles of ArsR family members in Brucella remain obscure. Here, we show that ArsR6 (BSS2_RS07325) is required for Brucella survival both under heat, oxidative, and osmotic stress and in a murine infection model in vivo. RNA-seq and ChIP-seq reveal that 34 potential target genes for ArsR6 protein were identified, among which eight genes were up-regulated and 26 genes were down-regulated, including outer membrane protein 25D (Omp25D). ArsR6 autoregulates its own expression to maintain bacterial intracellular Cu/Ni homeostasis to benefit bacterial survival in hostile environments. Moreover, ArsR6 also regulates the production of virulence factor Omp25D, which is important for the survival of Brucella under stress conditions. Significantly, Omp25D deletion strain attenuated in a murine infection model in vivo. Altogether, our findings reveal a unique mechanism in which the ArsR family member ArsR6 autoregulates its expression and also modulates Omp25D expression to maintain metal ion homeostasis and virulence in Brucella.

Brucella suis, the causative agent of brucellosis, poses a significant public health and animal husbandry threat. However, the role of the alanine racemase (alr) gene, which encodes alanine racemase in Brucella, remains unclear. Here, we analyzed an alr deletion mutant and a complemented strain of Brucella suis S2. The knockout strain displayed an unaltered, smooth phenotype in acriflavine agglutination tests but lacked the core polysaccharide portion of lipopolysaccharide (LPS). Genes involved in the LPS synthesis were significantly upregulated in the deletion mutant. The alr deletion strain exhibited reduced intracellular viability in the macrophages, increased macrophage-mediated killing, and upregulation of the apoptosis markers. Bcl2, an anti-apoptotic protein, was downregulated, while the pro-apoptotic proteins, Bax, Caspase-9, and Caspase-3, were upregulated in the macrophages infected with the deletion strain. The infected macrophages showed increased mitochondrial membrane permeability, Cytochrome C release, and reactive oxygen species, activating the mitochondrial apoptosis pathway. These findings revealed that alanine racemase was dispensable in B. suis S2 but influenced the strain’s rough features and triggered the mitochondrial apoptosis pathway during macrophage invasion. The deletion of the alr gene reduced the intracellular survival and virulence. This study enhances our understanding of the molecular mechanism underlying Brucella’s survival and virulence and, specifically, how alr gene affects host immune evasion by regulating bacterial LPS biosynthesis.