The (p)ppGpp synthetase Rsh promotes rifampicin tolerant persister cell formation in Brucella abortus by regulating the type II toxin-antitoxin module mbcTA.

Persister cells are transiently tolerant to antibiotics and are associated with recalcitrant chronic infections due to recolonization of host cells after antibiotic removal. Brucella spp. are facultative pathogens that establish intracellular infection cycles in host cells which results in chronic persistent infections. Brucella abortus forms multi-drug persister cells which are promoted by the (p)ppGpp synthetase Rsh during rifampicin exposure. Here, we confirmed that Rsh promoted persister cells formation in B. abortus stationary phase treated with rifampicin and enrofloxacin. Deletion of the gene for Rsh decreased persister cells level in the presence of these drugs in different growth phases. However, persister cells formation by deletion strain varied in different growth phases in the presence of other antibiotics. Rsh also was involved in persister cells formation during rifampicin treatment under certain stress conditions, including acidic conditions, exposure to PBS, and heat stress. Moreover, Rsh impacted persister cell levels during rifampicin or enrofloxacin treatment in RAW264.7 macrophages. Certain typeIItoxin-antitoxin modules were upregulated under various stress conditions in B. abortus. We established that Rsh positively regulated the type II toxin-antitoxin mbcTA. Moreover, rifampicin-tolerant persister cells formation was elevated and ATP levels were decreased when mbcTA promoter was overexpressed in Rsh deletion background in stationary phase. Our results establish that (p)ppGpp synthetase Rsh plays a key role in B. abortus persistence and may serve as a potent novel target in combination with rifampicin in the development of new therapeutic approaches and prevention strategies to treat chronic infections of Brucella.

Deletion of the alr gene in Brucella suis S2 attenuates virulence by enhancing TLR4-NF-κB-NLRP3- mediated host inflammatory responses.

Brucella is an intracellular parasitic bacterium lacking typical virulence factors, and its pathogenicity primarily relies on replication within host cells. In this study, we observed a significant increase in spleen weight in mice immunized with a Brucella strain deleted of the gene for alanine racemase (Alr), the enzyme responsible for alanine racemization (Δalr). However, the bacterial load in the spleen markedly decreased in the mutant strain. Concurrently, the ratio of white pulp to red pulp in the spleen was increased, serum IgG levels were elevated, but no significant damage to other organs was observed. In addition, the inflammatory response was potentiated and the NF-κB-NLRP3 signaling pathway was activated in macrophages (RAW264.7 Cells and Bone Marrow-Derived Cells) infect ed with the Δalr mutant. Further investigation revealed that the Δalr mutant released substantial amounts of protein in a simulated intracellular environment which resulted in heightened inflammation and activation of the TLR4-NF-κB-NLRP3 pathway in macrophages. The consequent cytoplasmic exocytosis reduced intracellular Brucella survival. In summary, cytoplasmic exocytosis products resulting from infection with a Brucella strain deleted of the alr gene effectively activated the TLR4-NFκB-NLRP3 pathway, triggered a robust inflammatory response, and reduced bacterial survival within host cells. Moreover, the Δalr strain exhibits lower toxicity and stronger immunogenicity in mice.

A potential virulence factor: Brucella flagellin FliK does not affect the main biological properties but inhibits the inflammatory response in RAW264.7 cells.

The bacterial flagellum is an elongated filament that protrudes from the cell and is responsible for bacterial motility. It can also be a pathogen-associated molecular pattern (PAMP) that regulates the host immune response and is involved in bacterial pathogenicity. In contrast to motile bacteria, the Brucella flagellum does not serve a motile purpose. Instead, it plays a role in regulating Brucella virulence and the host's immune response, similar to other non-motile bacteria. The flagellin protein, FliK, plays a key role in assembly of the flagellum and also as a potential virulence factor involved in the regulation of bacterial virulence and pathogenicity. In this study, we generated a Brucella suis S2 flik gene deletion strain and its complemented strain and found that deletion of the flik gene has no significant effect on the main biological properties of Brucella, but significantly enhanced the inflammatory response induced by Brucella infection of RAW264.7 macrophages. Further experiments demonstrated that the FliK protein was able to inhibit LPS-induced cellular inflammatory responses by down-regulating the expression of MyD88 and NF-κB, and by decreasing p65 phosphorylation in the NF-κB pathway; it also inhibited the expression of NLRP3 and caspase-1 in the NLRP3 inflammasome pathway. In conclusion, our study suggests that Brucella FliK may act as a virulence factor involved in the regulation of Brucella pathogenicity and modulation of the host immune response.

(P)ppGpp synthetase Rsh participates in rifampicin tolerance of persister cells in Brucella abortus in vitro.

Brucella abortus is facultative intracellular pathogen that causes chronic persistent infections and results in abortion and infertility in food animals. Recurrent infections can be one of the results of persister cells formation that transiently displays phenotypic tolerance to high dose of antibiotics treatment. We examined persister cells formation of B. abortus strain A19 in stationary phase and investigated a potential role for the (p)ppGpp synthetase Rsh in this process. We found that B. abortus stationary phase cells can produce higher levels of multi-drugs tolerant persister cells in vitro under high dose of antibiotics (20 × MIC) exposure than do exponential phase cells. Persister cell formation was also induced with environmental stressors pH 4.5, 0.01 M PBS (pH7.0), 2% NaCl and 25 °C, upon exposure to ampicillin, enrofloxacin and rifampicin. Persister cells were not formed following exposure to 1 mM H2O2. The numbers of persister cells were significantly increased following uptake of B. abortus stationary phase cells by RAW264.7 macrophages in contrast with cultures in TSB liquid medium. Environmental stressors to B. abortus significantly increased expression of rsh mRNA level. The rsh null mutant (Δrsh) formed significantly fewer persister cells than the complemented (CΔrsh) and wildtype (WT) strains under high dose of rifampicin in vitro. These data for the first time demonstrate that B. abortus can produce multi-drug tolerant persister cells in stationary phase. The (p)ppGpp synthetase Rsh is necessary for persister cell formation in B. abortus in the presence of rifampicin. On this basis, a new understanding of the recurrent infections of Brucella was advanced, thus provided a new basis for revelation of pathogenic mechanism of the chronic persistent infection in Brucella.

Alr Gene in Brucella suis S2: Its Role in Lipopolysaccharide Biosynthesis and Bacterial Virulence in RAW264.7.

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.

Brucella BtpB Manipulates Apoptosis and Autophagic Flux in RAW264.7 Cells.

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.

BtpB inhibits innate inflammatory responses in goat alveolar macrophages through the TLR/NF-κB pathway and NLRP3 inflammasome during Brucella infection.

Brucella species are infectious facultative intracellular pathogens. They have evolved multiple strategies to thwart immune responses and replicate in macrophages for chronic persistence in the host. As a Brucella effector, BtpB is transferred into target cells through the type IV secretion system. BtpB, a Toll/interleukin-1 receptor domain-containing protein, blocks host innate immune responses by interfering with Toll-like receptor signaling. However, the intracellular targets and their activated downstream pathways remain unclear. In this study, we constructed a strain of Brucella suis S2 with a deletion in the gene for BtpB, ΔbtpB, and the complemented strain, C-ΔbtpB with a restored copy of the btpB gene. The bacterial growth curves and stress resistance results showed that BtpB did not affect B. suis S2 growth. Infection of alveolar macrophages with WT and ΔbtpB strains showed that BtpB inhibited TLR2 and TLR4 expression and attenuated NLRP3 inflammasome activation. BtpB also attenuated secretion of the Brucella-induced proinflammatory cytokines, IL-1ß, IL-6, and TNF-α, in alveolar macrophages while up-regulating IL-10 expression. In general, the results confirmed that BtpB specifically inhibits TLR2/TLR4 and disrupts NLRP3 signaling pathways to inhibit host immune responses in early Brucella infections.