| Brucella spp.are facultative intracellular Gram-negative bacteria which develop a disease called brucellosis.Every year,Brucella infection cause enormous economic losses to the livestock industry all over the world.Meanwhile,Brucella melitensis,Brucella abortus and Brucella suis as important pathogens of zoonosis seriously threaten human health and life.When hosts were infected by Brucella,the bacteria were phagocytosed by phagocytes and undergone the adaptation period from the bacterial entry to initiating replication.Around 10% of intracellular Brucella could be survived during the adaptation period.After 12 hours of engulfment by phagocytes,Brucella started intracellular replication and then were released outside the cells in the form of autophagic vesicles from 48 to 72 hours after infection.Long non-coding RNAs(lncRNAs)are transcripts more than 200 bp in length and without protein coding potential.Currently,profiling the differentially expressed lncRNAs from macrophages RAW264.7during the period of Brucella abortus infection was poorly reported.By employing genome-wide transcriptome sequencing,here we identified 8,6,28,130,and 94 differentially expressed lncRNAs in RAW264.7 cells resulted from infection of a smooth Brucella abortus strain 2308(S2308)at 4,8,12,24,and 48 hours post-infection(hpi),respectively;20,40,181,6 and 173 differentially expressed lncRNAs caused by infection of a rough Brucella abortus Δrfb E at 4,8,12,24 and 48 hpi,respectively.GO analysis results showed lncRNA target genes in the early stage of infection were widely involved in cellular responses on interferon,tumor necrosis factor,interleukin and other cytokines as well as inflammatory,and chemotaxis of monocyte,lymphocyte and natural killer cell.Furthermore,some lncRNA target genes were related to response against LPS stimulus.However,many lncRNA target genes are associated with the metabolism and biological synthesis of macrophages at the later stage of infection,according to the GO annotation.In addition,results of GO annotation with cellular components displayed multiple lncRNA target genes were localized in membrane-related organelles while reports revealed membranerelated organelles play an important role in Brucella infection.In KEGG analysis,lncRNA target genes were connected to immune-related signals pathway such as tumor necrosis factor signaling pathway,NOD-like receptor signaling pathway,Toll-like receptor signaling pathway,NF-κB signaling pathway,cytokine and cytokine receptor interaction,as well as other pathways like Jak-STAT signaling pathway,Fc gamma R-mediated cell phagocytosis and bacterial invasion.Moreover,pathways of pathogen infection,metabolism and biological synthesis of macrophages were enriched during the Brucella infection.In this study,in total 185 differentially expressed lncRNAs resulted from Brucella infection were verified by RT-PCR and Sanger sequencing.Subsequently,results of qRT-PCR showed that the number of differentially expressed lncRNAs(fold change > 2)induced by S2308 were 2,3,6,30,and 32 at 4,8,12,24,and 48 hpi,respectively;the number of differentially expressed lncRNAs(fold change > 2)induced by Δrfb E were 8,8,49,1,and 43 at 4,8,12,24,and 48 hpi,respectively.In S2308 infected macrophages,the differential expressions of 12 lncRNAs were dependent on the infectious dose of S2308,and these lncRNAs had higher levels of differential expression at 24 and 48 hpi.In Δrfb E infected macrophages,qRT-PCR results revealed the expression of 6 lncRNAs were very obviously differential from 12 to 48 hpi(p < 0.05).Moreover,expression of these 6 lncRNA target genes at m RNA levels were significantly changed in macrophages during the infection of S2308 and Δrfb E,respectively(p < 0.05).In addition,expression of 18 differentially expressed lncRNAs screened from analyzed RNA-seq data of S2308-infected macrophages were significantly altered in the spleens of S2308-infected mice,and 12 of them were significantly abnormally expressed in the livers(p < 0.05).In order to investigate the transcription of lncRNAs,5’ end of ENSMUST00000186844.1 and LNC＿000428 were characterized by 5’ RACE to confirm the transcription start site(TSS),followed by knocking down their expression in RAW264.7 cells with CRISPR-mediated interference.Knockdown of ENSMUST00000186844.1 increased the colony forming units(CFUs)of S2308 in RAW264.7 cells,and decreased the expression of PHLDA1 which is the antisense target gene of the lncRNA.Similarly,interfering the expression of LNC＿000428 also raised the CFUs of S2308 in RAW264.7 cells,and downregulated expression of TNFRSF8 and TNFRSF1 B,which are antisense and upstream target genes of LNC＿000428,respectively.TSS of PHLDA1 were also determined by 5’ RACE and translation initiation site of this gene were predicted.When expression of PHLDA1 in RAW264.7 cells were knocked out by CRISPR,intracellular CFUs of S2308 were increased.In summary,a number of differentially expressed lncRNAs in macrophages were described by applying genome-wide transcriptome during the infection of smooth and rough Brucella abortus from the early to late stages.Enrichment of GO and KEGG analysis showed that the differentially expressed lncRNA target genes were related to biological processes of macrophages,such as cellular immune response,pathogen infection,cell metabolism and synthesis.A number of differentially expressed lncRNAs from RNA-seq data were verified by RT-PCR,Sanger sequencing and qRT-PCR.The differential expression levels of several lncRNAs in S2308-infected RAW264.7 cells were dependent on the time and dose of Brucella infection.In addition,some lncRNAs were differentially expressed in mice infected with S2308.After knockdown of ENSMUST00000186844.1 and LNC＿000428 in macrophages increased the number of intracellular Brucella,respectively.Moreover,knockout of the expression of PHLDA1 which is antisense target gene of ENSMUST00000186844.1 were multiplied CFUs of S2308 in RAW264.7 cells.These results showed macrophages utilized lncRNAs to control the intracellular Brucella.Therefore,this study can provide a foundation for further investigation of more lncRNAs correlated Brucella infection in macrophages. |
