The Transcriptome Analysis Of Brucella By RNA-seq And Function Study Of SRNA

Brucella species are typical zoonotic facultative intracellular pathogens, can cause infectiousAbortions, placentitis, epididymitis and orchitis in cattle, sheep, goats, swine and Malta Fever inman. Brucellosis is one of the world’s most widespread zoonotic disease. Brucella infection occursthrough inhalation or ingestion of organisms via the nasal, oral, and pharyngeal cavities. Thebacteria are phagocytosed by macrophages, stay in membran-bound vacuole.rly endosomes(Brucella-containing vacuole, BCV). More than90%of Brucella are killed and Less than10%Brucella are survived in early phagosome. Replicative phagosomes (Brucellosome) provide anintracellular niche for Brucella growth, and Lysosomes do not fuse with the Brucella phagosome.Brucellosome is a niche of nutrient depletion, acidification, and hypoxia, providing Brucella witha harsh environment. Therefore, the intracellular survival is the most important virulencecharacteristics. In the present study, the transcriptome analysis of Brucella differential expressiongenes under macrophages analogue of nutrient stress in vitro and normal culture was investagatedvia RNA-seq to reveal the molecular mechanism of virulence, pathogenicity, and metabolismrelated to nutrient stress. sRNAs are important regulators of bacterial adaptation to environmentalstresses, and play important roles in gene expression regulations. Meanwhile, prediction,identification of Brucella sRNAs via transcriptome analysis and bioinformatics were done tosearch sRNA adapting the macrophages intracellular conditions of nutrient depletion, acidification,hypoxia, and reveal the important role of sRNAs in Brucella intracellular survival and intracellularcompetition.Brucella melitensis16M were cultured under nutrient stress GEM7.0and normal cultureTSB7.0, RNA extraction, RNA sequencing and transcriptome analysis of Brucella differentialexpression genes. Compared with TSB7.0,689genes in GEM7.0were differentially expressedwith a difference higher than twoflod. A total of308genes were upregulated and381genes weredownregulated. The genes associated with first stage fatty acid metabolism were upregulated, andThe genes associated with second stage fatty acid metabolism were downregulated, indicating thatfatty acid metabolism were under nutrient stress condition. Pathogenesis and Bacterial secretionsystem under nutrient stress condition were upregulated; nutrient stress and pathogenesis are twocoordinated characteristics of Brucella. The results that T4SS system coding genes under nutrientstress condition were enriched with high expression further confirmed T4SS system plays animportant role in Brucella resisting external adverse environment. Genes associated with OmpRSignal transduction system under nutrient stress condition were upregulated, indicating that OmpRSignal transduction system is one important component in response to nutrient stress. Genesassociated with ABC transport system Importers and Exporters were downregulated, indicatingthat ABC transport systems were downregulated and Brucella may be smooth type LPS structureinto the rough under nutrient stress.RNA-seq reads were assembled and Brucella sRNAs were discovered through bioinformaticspredictions. Peaks were located on intergenic regions(IGRs) between two annotated genes.Terminators were identified within200nt downstream, and promoters were indentified within200nt upstream. The strand-specific RNA-seq analysis of transcriptome of16M indicated that a totalof48new putative sRNA with promoter and terminator were transcribed,23in the sense strand and25in the antisense strand. Meanwhile, A total of21sRNA were predicted by Bioinformaticstranscripts prediction,15in the sense strand and6in the antisense strand. Northern blot results ofDIG-labeled RNA probes hybridization were shown that15of21sRNA predicted byBioinformatics transcripts prediction were transcribed. Most of sRNA were encoded on intergenicregions of bacteria genome, which could be cotranscribed with upstream and/or downstream ORFs.Of15sRNA verified by Norhtern blot,6were oriented in the opposite direction from the twoflanking ORFs, indicating that this putative sRNA should be transcribed independently of theflanking ORFs.9sRNA could be cotranscribed with upstream and/or downstream ORFs. Theresults were shown that7sRNA were cotranscribed with upstream and/or downstream ORFs, andBSR5and BSR8were transcribed independently of the flanking ORFs.To test the functions of independently transcribed BSR16and BSR17, the full length ofBSR16and BSR17were determined by5’ RACE and3’ RACE. The transcription start site ofBSR16and BSR17were635852and1187596. The transcription end site of BSR16and BSR17were636137and1187668. Then, the deletion mutant and overexpression strains of BSR16andBSR17were constructed, and the intracellular survival phenotypes were compared. The results ofthe growth curve were shown that16M grow fast than16M-△B SR16,16M-BSR16,16M-△BSR17and16M-BSR17, and reveals that BSR16and BSR17are associated with Brucellagrowth and metabolism. Meanwhile, the survival experents of Brucella under High salt, highosmolarity, oxidative stress, low pH and heat stress conditions in vitro revealed that the survivalrate of16M-BSR16was lower than16M and16M-△B SR16, and the overexpression of BSR16reduced the survival rate of Brucella in macrophages’ intracellular environments and decreased theresistant ability with various pressures. The survival rate of16M-△BSR17and16M-BSR17weremuch lower than16M under various stress conditions. The deletion and overexpression of BSR17reduced the ability to decreased the survival rate of Brucella under various stress conditions,reduced the ability to resist the various pressures, such as the intracellular oxidative stress andacid-base environment. After Brucella are transported within phagocytic cells, the number ofintracellular bacteria are related to incursion and viability. The results of mouse virulence survivalwere shown that the competive advantage of16M is more large than16M-△B SR16, but notobvious. The competitive advantage of16M-△BSR17and16M-BSR17decreased verysignificantly. The deletion and overexpression of BSR17reduced the ability of Brucella adaptionto host spleen cells, suggesting that BSR17plays an important role in long-term survival ofBrucella in the spleen cells and the corect expression of BSR17is needed for the host long-termsurvival of Brucella. To survive in host, Brucella need to overcome or resist variousbactericidal/bacteriostatic environments of host, such as nutrient stress of BCVs. The results ofBrucella long-term survival under nutrient stress condition in vitro were shown that the deletionand overexpression of BSR16and BSR17declined the viability, and the decline of BSR16andBSR17overexpression strain was more obvious. Overexpression of BSR16and BSR17significantly reduced the ability of Brucella adaption to nutrient stress condition.Bacterial sRNAs act as key players in posttranscriptional regulation through base pairingwith target mRNAs. Therefore, searching target genes regulated by sRNA is the key to study thesRNA mechanism. Firstly, Target genes of BSR16and BSR17were analyzed throughbioinformatics predictions. The results showed that BSR16target genes were associated withtransport and metabolism, transcription, posttranslational modification, Intracellular transport, cellmembrane biogenesis and energy production and conversion. BSR17target genes were associatedwith material transport and metabolism, replication&recombination&repair, and signaltransduction mechanisms. Moreover, several target genes were associated with Brucella intracellular survival. In most cases, the RNA chaperone protein Hfq is requried to stabilize thesRNA-mRNA interaction. Therefore, the relationship between BSR16and Hfq were investigated.The results showed that BSR16relies on Hfq for its expression, and the expression of BSR16in16M-△hfq decreased significantly. To further analyze the function of BSR16, we compareddifferetially expressed proteomics analysis of16M and16M-BSR16, finding the target proteins ofBSR16. The results of comperative proteome and MALDI-TOF-MS of16M and16M-BSR16（BSR16overexpression）showed that, proteins were assoiciated with amino acid transport andmetabolism, Energy production and conversion, carbohydrate transport and metabolism,carbohydrate transport and metabolism, translation&ribosomal structure and biogenesis, cellwall/membrane/envelope biogenesis, posttranslational modification&protein turnover&chaperones, signal transduction mechanisms, lipid transport and metabolism were differentioniallyexpressed. All these data present that BSR16controls relevant phenotypes by regulating proteinsassociated with intracellular survival and virulence. These results above indicated that there are acertain number of sRNAs in Brucella, and sRNAs play an important role in contributing adaptionto intracellular survival of Brucella.These finding of the transcriptome and DEG(differential expression genes) of Brucella undernutrient stress condition expand our knowledge of molecular mechanism of virulence,pathogenesis and metabolism under nutrient stress condition. The global predication,indentification of Brucella sRNAs via transcriptome analysis and bioinformatics, and the approachof the roles of sRNAs in adapation of Brucella to hostile environment in the host cell, providevarious important clues for understanding of the molecular mechanism of intracellular survival ofBrucella, sRNA in resistance to environmental stimuli.