ObjectivesThe 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 methodsMicroarray 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.ResultsThe 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-1β 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.ConclusionsOur 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.

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.

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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 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.