Systemic Pharmacology Reveals the Potential Targets and Signaling Mechanisms in the Adjuvant Treatment of Brucellosis with Traditional Chinese Medicine.

Human brucellosis is one of the world's most common zoonoses, caused by Brucella infection and characterized by induced inflammation, which in severe cases can lead to abortion and sterility in humans and animals. There is growing evidence that traditional Chinese medicine (TCM) is beneficial as an adjunct to the treatment of brucellosis. However, its specific targets of action and molecular mechanisms remain unclear. In this study, a systematic pharmacological approach was applied to demonstrate pharmacological targets, biological functions, and signaling pathways of TCM as an adjunct to the treatment of brucellosis (TCMTB). The results of network pharmacology were further verified by in vitro experiments. Network analysis revealed that 133 active ingredients and 247 targets were screened in TCMTB. Further data analysis identified 21 core targets and 5 core compounds in TCMTB, including beta-sitosterol, quercetin, kaempferol, luteolin, and paeoniflorin. Gene ontology and the Kyoto Encyclopedia of Gene and Genome analysis showed that TCMTB might actively treat brucellosis by regulating inflammatory response, enhancing immune function, and targeting signaling pathways such as tuberculosis and TNF. Molecular docking results showed that multiple compounds could bind to multiple targets. Further, in vitro experiments confirmed that quercetin, among the active compounds screened, induced the strongest immunomodulatory and pro-inflammatory cytokine production during Brucella abortus infection. Further, quercetin induced nitric oxide production, which attenuated the ability of B. abortus to internalize THP-1 cells as well as intracellular survival. This study reveals the mechanism by which TCMTB aids in the treatment of brucellosis through a synergistic multicomponent, multipathway, and multitarget action. The contribution of quercetin treatment to B. abortus infection was demonstrated for the first time, which may be related to the quercetin-induced production of nitric oxide and immunomodulatory and inflammatory cytokines. These predictions of the core compounds and targets may be used in the future for the clinical treatment of brucellosis.

Brucella abortus modulates macrophage polarization and inflammatory response by targeting glutaminases through the NF-κB signaling pathway.

Objectives: The mechanism of Brucella infection regulating macrophage phenotype has not been completely elucidated until now. This study aimed to determine the mechanism of Brucella abortus in the modulation of macrophage phenotype using RAW264.7 cells as a model. Materials and methods: RT-qPCR, ELISA and flow cytometry were used to detect the inflammatory factor production and phenotype conversion associated with M1/M2 polarization of macrophages by Brucella abortus infection. Western blot and immunofluorescence were used to analyze the role of nuclear factor kappa B (NF-κB) signaling pathway in regulation of Brucella abortus-induced macrophage polarization. Chromatin immunoprecipitation sequencing (Chip-seq), bioinformatics analysis and luciferase reporter assay were used to screen and validate NF-κB target genes associated with macrophage polarization and further verify its function. Results: The results demonstrate that B. abortus induces a macrophage phenotypic switch and inflammatory response in a time-dependent manner. With the increase of infection time, B. abortus infection-induced M1-type increased first, peaked at 12 h, and then decreased, whereas the M2-type decreased first, trough at 12 h, and then increased. The trend of intracellular survival of B. abortus was consistent with that of M2 type. When NF-κB was inhibited, M1-type polarization was inhibited and M2-type was promoted, and the intracellular survival of B. abortus increased significantly. Chip-seq and luciferase reporter assay results showed that NF-κB binds to the glutaminase gene (Gls). Gls expression was down-regulated when NF-κB was inhibited. Furthermore, when Gls was inhibited, M1-type polarization was inhibited and M2-type was promoted, the intracellular survival of B. abortus increased significantly. Our data further suggest that NF-κB and its key target gene Gls play an important role in controlling macrophage phenotypic transformation. Conclusions: Taken together, our study demonstrates that B. abortus infection can induce dynamic transformation of M1/M2 phenotype in macrophages. Highlighting NF-κB as a central pathway that regulates M1/M2 phenotypic transition. This is the first to elucidate the molecular mechanism of B. abortus regulation of macrophage phenotype switch and inflammatory response by regulating the key gene Gls, which is regulated by the transcription factor NF-κB.

Survival characteristics and transcriptome profiling reveal the adaptive response of the Brucella melitensis 16M biofilm to osmotic stress.

Brucella can inhabit hostile environments, including osmotic stress. How Brucella responds collectively to osmotic stress is largely unexplored, particularly in spatially structured communities such as a biofilm. To gain insight into this growth mode, we set out to characterize the Brucella melitensis 16M biofilm, describe its phenotype, and carry out a comparative transcriptomic analysis between biofilms under osmotic stress and control conditions. We determined that the bacteria challenged with 1.5 M NaCl had a reduced ability to aggregate and form clumps and develop a biofilm; however, the salt stress promoted the release of the outer membrane vesicles from the biofilm. Together with the genotypical response to osmotic stress, we identified 279 differentially expressed genes in B. melitensis 16M grown under osmotic conditions compared with control conditions; 69 genes were upregulated and 210 downregulated. Under osmotic stress, the main changed genes of biofilm were predicted to be involved in flagellar assembly, cell envelope, translation, small RNA regulation, transport and binding proteins, and energy metabolism. In addition, the ABC transporter was enriched in the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. We highlight 12 essential ABC transporter genes associated with a bacterial response to osmotic stress at the biofilm stage, including one specific locus, BME_RS12880, mediating betaine accumulation in biofilms to eliminate osmotic stress. The current study results can help researchers gain insights into B. melitensis 16M biofilm adaptation to osmotic stress and provide information for developing intervention strategies to control Brucella.

The Flagellar Transcriptional Regulator FtcR Controls Brucella melitensis 16M Biofilm Formation via a betI-Mediated Pathway in Response to Hyperosmotic Stress.

The expression of flagellar proteins in Brucella species likely evolved through genetic transference from other microorganisms, and contributed to virulence, adaptability, and biofilm formation. Despite significant progress in defining the molecular mechanisms behind flagellar gene expression, the genetic program controlling biofilm formation remains unclear. The flagellar transcriptional factor (FtcR) is a master regulator of the flagellar system's expression, and is critical for B. melitensis 16M's flagellar biogenesis and virulence. Here, we demonstrate that FtcR mediates biofilm formation under hyperosmotic stress. Chromatin immunoprecipitation with next-generation sequencing for FtcR and RNA sequencing of ftcR-mutant and wild-type strains revealed a core set of FtcR target genes. We identified a novel FtcR-binding site in the promoter region of the osmotic-stress-response regulator gene betI, which is important for the survival of B. melitensis 16M under hyperosmotic stress. Strikingly, this site autoregulates its expression to benefit biofilm bacteria's survival under hyperosmotic stress. Moreover, biofilm reduction in ftcR mutants is independent of the flagellar target gene fliF. Collectively, our study provides new insights into the extent and functionality of flagellar-related transcriptional networks in biofilm formation, and presents phenotypic and evolutionary adaptations that alter the regulation of B. melitensis 16M to confer increased tolerance to hyperosmotic stress.

Molecular Detection of Zoonotic and Veterinary Pathogenic Bacteria in Pet Dogs and Their Parasitizing Ticks in Junggar Basin, North-Western China.

Despite the recognized epidemiological importance of ticks as vectors for pathogens that cause numerous zoonotic and veterinary diseases, data regarding the pathogens of pet dogs and their parasitic ticks in the Junggar Basin are scarce. In this study, a total of 178 blood samples and 436 parasitic ticks were collected from pet dogs in Junggar Basin, Xinjiang Uygur Autonomous Region (XUAR), north-western China. All ticks were identified as Rhipicephalus turanicus sensu stricto (s.s.) according to morphological and molecular characteristics. Rh. turanicus s.s. ticks were collected from pet dogs in China for the first time. Seven tick-borne pathogens, such as Ehrlichia chaffeensis, Anaplasma phagocytophilum, Rickettsia massiliae, Candidatus R. barbariae, Brucella spp., Rickettsia sibirica, and Anaplasma ovis, were detected from ticks, whereas the first five bacteria were detected from blood samples of dogs. Brucella spp. was the most predominant pathogen in both blood samples and ticks of pet dogs, with the detection rates of 16.29 and 16.74%, respectively. Moreover, 17 ticks and 1 blood sample were co-infected with two pathogens, and 1 tick was co-infected with three pathogens. This study provided molecular evidence for the occurrence of Anaplasma spp., Ehrlichia spp., Rickettsia spp., and Brucella spp. circulating in pet dogs and their parasitic ticks in Junggar Basin, north-western China. These findings extend our knowledge of the tick-borne pathogens in pet dogs and their parasitic ticks in Central Asia; therefore, further research on these pathogens and their role in human and animal diseases is required.

Corrigendum: Brucella-Induced Downregulation of lncRNA Gm28309 Triggers Macrophages Inflammatory Response Through the miR-3068-5p/NF-κB Pathway.

[This corrects the article DOI: 10.3389/fimmu.2020.581517.].

Functional analysis of Bucella reveals transcriptional regulation of MarR.

Brucellosis is a zoonotic infectious disease caused by Brucella infection. MarR-family transcription factors are closely related to diverse physiological functions necessary for many pathogens adaptation to environmental changes. However, whether the MarR-family transcription factors are involved in virulence, mediated inflammatory responses and regulated virulence gene expression in the intracellular pathogen Brucella are still unknown. Therefore, we created a 2308ΔMarR6 mutant of B. abortus 2308 (S2308). Virulence and inflammatory cytokines assays were performed using a murine macrophage cell line (RAW 264.7). We also performed chromatin immunoprecipitation of MarR6 followed by next-generation sequencing (ChIP-seq). The results showed that 2308ΔMarR6 was significantly reduced survival capability in RAW 264.7. After the macrophages were infected with 2308ΔMarR6, the levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), interleukin-12 (IL-12), interferon-gamma (IFN-γ) and macrophage chemoattractant protein-1 (MCP-1) were decreased and were significantly lower than that for the S2308-infected group, indicating that the 2308ΔMarR6 mutant could reduce the secretion of inflammatory cytokines. Furthermore, we detected 122 intergenic ChIP-seq peaks of MarR6 binding distributed across the Brucella genome. Taken together, the research has recorded valuable data about MarR6. Our findings are of great significance in elucidating the function of MarR6.

Brucella-Induced Downregulation of lncRNA Gm28309 Triggers Macrophages Inflammatory Response Through the miR-3068-5p/NF-κB Pathway.

Objectives: The underlying mechanism of the inflammatory response against Brucellosis caused by Brucella remains poorly understood. This study aimed to determine the role of long non-coding RNAs (lncRNAs) in regulating of inflammatory and anti-Brucella responses. Materials and methods: Microarray analysis was performed to detect differentially expressed lncRNAs in THP-1 cells infected with an S2308 Brucella strain. The candidate lncRNAs were screened using bioinformatic analysis and siRNAs; bioinformatic prediction and luciferase reporter assay were also conducted, while inflammatory responses was assessed using RT-qPCR, western blot, immunofluorescence, ELISA, HE, and immunohistochemistry. Results: The lncRNA Gm28309 was identified to be involved in regulating inflammation induced by Brucella. Gm28309, localized in the cytoplasm, was down-expressed in RAW264.7 cells infected with S2308. Overexpression of Gm28309 or inhibition of miR-3068-5p repressed p65 phosphorylation and reduced NLRP3 inflammasome and IL-1ß and IL-18 secretion. Mechanistically, Gm28309 acted as a ceRNA of miR-3068-5p to activate NF-κB pathway by targeting κB-Ras2, an inhibitor of NF-κB signaling. Moreover, the number of intracellular Brucella was higher when Gm28309 was overexpressed or when miR-3068-5p or p65 was inhibited. However, these effects were reversed by the miR-3068-5p mimic. Conclusions: Our study demonstrates, for the first time, that LncRNAs are involved in regulating immune responses during Brucella infection, and Gm28309, an lncRNA, plays a crucial role in activating NF-κB/NLRP3 inflammasome signaling pathway.