布鲁氏菌转录组测序分析及sRNA功能研究
摘要
布鲁氏菌是典型的人畜共患胞内寄生菌，牛、羊、猪等家畜感染后能引起流产和不孕，人感染布病引起关节炎、地中海热等。布鲁氏菌病在世界各地广泛流行，危害严重。布鲁氏菌通过呼吸道、消化道和皮肤黏膜等感染宿主，进入宿主后被宿主巨噬细胞吞噬，与巨噬细胞膜相互作用形成BCV吞噬小体，约有10%的布鲁氏菌存活下来。布鲁氏菌在BCV吞噬小体内面临营养胁迫、低pH、低氧等苛刻环境压力，但吞噬小体内同时也为布鲁氏菌提供了一个逃避细胞吞噬、清除的内环境。因此，胞内生存是布鲁氏菌致病的关键环节。本研究通过对体外模拟巨噬细胞内营养胁迫条件与正常实验室培养条件下的布鲁氏菌转录组进行测序，比较分析营养胁迫条件下布鲁氏菌基因表达的变化，揭示与营养胁迫条件相关的毒力、致病、代谢等分子机制，进而为布鲁氏菌的胞内生存机制研究奠定基础。细菌非编码小RNA（non-coding small RNA，sRNA）是近年来在细菌中发现的一类RNA调控子，在应对环境变化的基因表达调控中发挥重要作用，是细菌适应环境压力的重要调控子。本研究通过转录组测序分析营养胁迫条件下的布鲁氏菌sRNA，同时结合生物信息学预测sRNA，对得到的sRNA进行功能分析，寻找与布鲁氏菌适应胞内压力环境相关的sRNA，揭示sRNA在布鲁氏菌胞内生存中的调控作用。
分别在营养胁迫培养基GEM7.0与标准培养基TSB7.0中培养布鲁氏菌标准强毒株16M，提取总RNA进行转录组测序（RNA-seq）。对测序结果进行分析，结果显示，与正常实验室培养条件（TSB7.0）相比，布鲁氏菌16M在营养胁迫条件下，大量基因表达发生明显的改变。3199个基因中，差异表达的基因有689个（21.54%），其中上调表达的基因有308个（9.63%），下调381个（11.91%）。在营养胁迫条件下，参与脂肪酸第一阶段代谢关键酶富集性上调表达，参与脂肪酸第二阶段代谢相关酶都富集性下调表达，表明营养胁迫条件下布鲁氏菌的脂肪酸代谢过程受到了影响。营养胁迫条件下，致病机制与细菌分泌系统相关基因广泛上调，表明营养胁迫与细菌致病性是布鲁氏菌紧密协作（coordinated）的两个特性。营养胁迫条件下，T4SS系统相关基因富集性高表达，进一步证实T4SS对于布鲁氏菌抵抗外界不良环境具有重要作用；OmpR以及信号转导反应调控基因上调，暗示该通路是布鲁氏菌适应营养胁迫的重要组成；O抗原输出系统基因表达水平下调，暗示布鲁氏菌在营养胁迫情况下可能导致光滑型LPS的结构转化成粗糙型。
对RNA-Seq方法获得的原始测序结果进行拼装和生物信息学分析寻找布鲁氏菌的sRNA。首先筛选位于基因间区，与邻近基因距离大于20nt，表达量与邻近基因相差15%的peak为候选peak。选取候选peak中高丰度表达的peak，向上游延伸200nt预测转录启动子，向下游延伸200nt预测转录终止子。从其中筛选出具有启动子和终止子的区域为sRNA的表达转录区域。通过筛选分析，最终得到48个sRNA，其中23个位于正链上，25个位于负链上。同时，我们通过转录单元预测法还预测出21个sRNA分子，其中15个位于正义链上，6个位于反义链上。利用northern blot对转录单元预测法预测出的21个sRNA进行验证，结果表明共有15个sRNA存在转录本。由于sRNA多位于基因间区，有可能与邻近的基因存在共转录，我们对这15个Northern blot验证阳性的sRNA与邻近基因的共转录情况进行了分析。结果表明，其中6个sRNA与邻近的上下游基因不在一条链上，肯定是单独转录的；而剩余的9个sRNA则通过RT-PCR的方法进行了验证，结果表明仅BSR5与BSR8与邻近基因不存在共转录，是单独转录的。
对单独转录的BSR16和BSR17进行了进一步的研究，首先通过5’RACE与3’RACE寻找BSR16与BSR17的转录起点与转录终点。实验结果表明，BSR16的转录起点为635852，转录终点为636137；BSR17的转录起点为1187596，转录终点为1187668。为了分析BSR16和BSR17的功能，我们构建了BSR16和BSR17的缺失株和过表达株，并对野生株、缺失株和过表达株的相关表型进行了比较分析，主要包括：生长曲线、体外刺激条件下的生存能力、饥饿实验和小鼠体内生存能力等。从生长曲线可以看出，当BSR16或BSR17缺失和过表达时，布鲁氏菌的生长速度减慢，提示BSR16、BSR17与布鲁氏菌的生长代谢相关。巨噬细胞内生存和繁殖是布鲁氏菌致病的关键，巨噬细胞内环境中包含多种杀（抑）菌物质，而布鲁氏菌必须适应这种环境才能在胞内生存和繁殖。因此，我们又分析BSR16和BSR17的缺失株和过表达株在模拟巨噬细胞内环境的多种体外胁迫条件下的生存能力，包括：高盐、高渗透压、氧化应激、酸、热等。实验结果表明，与16M野生株相比，BSR16过表达株16M-BSR16在各种体外胁迫条件下的生存率明显降低，而BSR16缺失突变株仅在高热压力下生存率下降。结果提示，BSR16的过表达可能导致布鲁氏菌抵抗外界环境压力的能力减弱。对于BSR17，在高盐、高热和氧压力条件，BSR17的缺失株和过表达株的生存率均明显降低。而在高渗条件下，仅BSR17突变株的生存率下降；在低pH值条件，仅过表达株的生存率下降。营养胁迫条件是布鲁氏菌在巨噬细胞内生存时遇到的重要环境压力，因此，我们又分析了BSR16和BSR17的缺失株和过表达株在营养胁迫条件下的长期生存能力。实验结果表明，BSR16和BSR17过表达时，布鲁氏菌在营养胁迫条件下的生存能力均明显减弱。为了进一步明确BSR16和BSR17与布鲁氏菌胞内存活能力的关系，我们对BSR16和BSR17的缺失株和过表达株在小鼠体内的生存能力进行了比较分析。实验结果表明，BSR16和BSR17的缺失与过表达均影响了布鲁氏菌在小鼠脾脏细胞内的生存能力，提示BSR16和BSR17的正确表达对于布鲁氏菌在小鼠体内的长期生存是必须的。
细菌的sRNA主要通过靶标mRNAs来发挥转录后调控作用，因此，寻找受sRNA调控的靶基因是研究sRNA作用机制的关键。我们首先通过生物信息学对BSR16和BSR17的靶基因进行了预测分析。结果表明，BSR16的靶基因主要涉及物质的转运和代谢、转录、转录后修饰、胞内运输、细胞膜合成、能量产生与转化等；BSR17的靶基因主要涉及物质转运和代谢基因、复制、基因重组和修复、信号转导机制等。其中，多个靶基因与布鲁氏菌的胞内生存能力有关。由于大多细菌sRNA与靶标mRNA的结合依赖于RNA分子伴侣Hfq，因此，我们分析了BSR16与Hfq的相关性。实验结果表明，BSR16与Hfq密切相关，是Hfq依赖性sRNA，当Hfq缺失后，BSR16的表达明显降低。为进一步分析BSR16的功能，我们利用双向电泳-质谱的蛋白质组学研究方法比较了16M和BSR16过表达株的全菌蛋白表达的差异，从蛋白质组水平鉴定BSR16调控的靶基因。研究结果表明，当BSR16过表达后，布鲁氏菌的蛋白表达发生了明显的变化。这些差异表达的蛋白主要涉及：物质转运和代谢、能源产生和转换、转录、翻译、翻译后修饰、细胞膜合成、分子伴侣和信号转导机制等。其中很多与布鲁氏菌的胞内生存和环境压力适应相关。BSR16过表达后，这些靶基因的表达发生改变，最终影响了布鲁氏菌对环境压力的适应能力和胞内生存能力。以上研究结果表明布鲁氏菌内存在一定数量的sRNA，而且sRNA在布鲁氏菌抵抗与适应巨噬细胞内恶劣环境中发挥重要作用。
本研究通过对布鲁氏菌在营养胁迫条件下的转录组测序、差异基因表达分析，对布鲁氏菌在营养胁迫条件下的毒力、致病、代谢等分子机制有了一定的认识。结合转录组测序和生物信息学预测对布鲁氏菌的全基因组进行扫描分析，寻找布鲁氏菌的sRNA，并对sRNA在布鲁氏菌适应环境压力和胞内生存中发挥的作用及其调控的靶基因进行了详细的探讨，为研究布鲁氏菌抵抗环境压力及胞内生存机制提供了重要线索。
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.
分别在营养胁迫培养基GEM7.0与标准培养基TSB7.0中培养布鲁氏菌标准强毒株16M，提取总RNA进行转录组测序（RNA-seq）。对测序结果进行分析，结果显示，与正常实验室培养条件（TSB7.0）相比，布鲁氏菌16M在营养胁迫条件下，大量基因表达发生明显的改变。3199个基因中，差异表达的基因有689个（21.54%），其中上调表达的基因有308个（9.63%），下调381个（11.91%）。在营养胁迫条件下，参与脂肪酸第一阶段代谢关键酶富集性上调表达，参与脂肪酸第二阶段代谢相关酶都富集性下调表达，表明营养胁迫条件下布鲁氏菌的脂肪酸代谢过程受到了影响。营养胁迫条件下，致病机制与细菌分泌系统相关基因广泛上调，表明营养胁迫与细菌致病性是布鲁氏菌紧密协作（coordinated）的两个特性。营养胁迫条件下，T4SS系统相关基因富集性高表达，进一步证实T4SS对于布鲁氏菌抵抗外界不良环境具有重要作用；OmpR以及信号转导反应调控基因上调，暗示该通路是布鲁氏菌适应营养胁迫的重要组成；O抗原输出系统基因表达水平下调，暗示布鲁氏菌在营养胁迫情况下可能导致光滑型LPS的结构转化成粗糙型。
对RNA-Seq方法获得的原始测序结果进行拼装和生物信息学分析寻找布鲁氏菌的sRNA。首先筛选位于基因间区，与邻近基因距离大于20nt，表达量与邻近基因相差15%的peak为候选peak。选取候选peak中高丰度表达的peak，向上游延伸200nt预测转录启动子，向下游延伸200nt预测转录终止子。从其中筛选出具有启动子和终止子的区域为sRNA的表达转录区域。通过筛选分析，最终得到48个sRNA，其中23个位于正链上，25个位于负链上。同时，我们通过转录单元预测法还预测出21个sRNA分子，其中15个位于正义链上，6个位于反义链上。利用northern blot对转录单元预测法预测出的21个sRNA进行验证，结果表明共有15个sRNA存在转录本。由于sRNA多位于基因间区，有可能与邻近的基因存在共转录，我们对这15个Northern blot验证阳性的sRNA与邻近基因的共转录情况进行了分析。结果表明，其中6个sRNA与邻近的上下游基因不在一条链上，肯定是单独转录的；而剩余的9个sRNA则通过RT-PCR的方法进行了验证，结果表明仅BSR5与BSR8与邻近基因不存在共转录，是单独转录的。
对单独转录的BSR16和BSR17进行了进一步的研究，首先通过5’RACE与3’RACE寻找BSR16与BSR17的转录起点与转录终点。实验结果表明，BSR16的转录起点为635852，转录终点为636137；BSR17的转录起点为1187596，转录终点为1187668。为了分析BSR16和BSR17的功能，我们构建了BSR16和BSR17的缺失株和过表达株，并对野生株、缺失株和过表达株的相关表型进行了比较分析，主要包括：生长曲线、体外刺激条件下的生存能力、饥饿实验和小鼠体内生存能力等。从生长曲线可以看出，当BSR16或BSR17缺失和过表达时，布鲁氏菌的生长速度减慢，提示BSR16、BSR17与布鲁氏菌的生长代谢相关。巨噬细胞内生存和繁殖是布鲁氏菌致病的关键，巨噬细胞内环境中包含多种杀（抑）菌物质，而布鲁氏菌必须适应这种环境才能在胞内生存和繁殖。因此，我们又分析BSR16和BSR17的缺失株和过表达株在模拟巨噬细胞内环境的多种体外胁迫条件下的生存能力，包括：高盐、高渗透压、氧化应激、酸、热等。实验结果表明，与16M野生株相比，BSR16过表达株16M-BSR16在各种体外胁迫条件下的生存率明显降低，而BSR16缺失突变株仅在高热压力下生存率下降。结果提示，BSR16的过表达可能导致布鲁氏菌抵抗外界环境压力的能力减弱。对于BSR17，在高盐、高热和氧压力条件，BSR17的缺失株和过表达株的生存率均明显降低。而在高渗条件下，仅BSR17突变株的生存率下降；在低pH值条件，仅过表达株的生存率下降。营养胁迫条件是布鲁氏菌在巨噬细胞内生存时遇到的重要环境压力，因此，我们又分析了BSR16和BSR17的缺失株和过表达株在营养胁迫条件下的长期生存能力。实验结果表明，BSR16和BSR17过表达时，布鲁氏菌在营养胁迫条件下的生存能力均明显减弱。为了进一步明确BSR16和BSR17与布鲁氏菌胞内存活能力的关系，我们对BSR16和BSR17的缺失株和过表达株在小鼠体内的生存能力进行了比较分析。实验结果表明，BSR16和BSR17的缺失与过表达均影响了布鲁氏菌在小鼠脾脏细胞内的生存能力，提示BSR16和BSR17的正确表达对于布鲁氏菌在小鼠体内的长期生存是必须的。
细菌的sRNA主要通过靶标mRNAs来发挥转录后调控作用，因此，寻找受sRNA调控的靶基因是研究sRNA作用机制的关键。我们首先通过生物信息学对BSR16和BSR17的靶基因进行了预测分析。结果表明，BSR16的靶基因主要涉及物质的转运和代谢、转录、转录后修饰、胞内运输、细胞膜合成、能量产生与转化等；BSR17的靶基因主要涉及物质转运和代谢基因、复制、基因重组和修复、信号转导机制等。其中，多个靶基因与布鲁氏菌的胞内生存能力有关。由于大多细菌sRNA与靶标mRNA的结合依赖于RNA分子伴侣Hfq，因此，我们分析了BSR16与Hfq的相关性。实验结果表明，BSR16与Hfq密切相关，是Hfq依赖性sRNA，当Hfq缺失后，BSR16的表达明显降低。为进一步分析BSR16的功能，我们利用双向电泳-质谱的蛋白质组学研究方法比较了16M和BSR16过表达株的全菌蛋白表达的差异，从蛋白质组水平鉴定BSR16调控的靶基因。研究结果表明，当BSR16过表达后，布鲁氏菌的蛋白表达发生了明显的变化。这些差异表达的蛋白主要涉及：物质转运和代谢、能源产生和转换、转录、翻译、翻译后修饰、细胞膜合成、分子伴侣和信号转导机制等。其中很多与布鲁氏菌的胞内生存和环境压力适应相关。BSR16过表达后，这些靶基因的表达发生改变，最终影响了布鲁氏菌对环境压力的适应能力和胞内生存能力。以上研究结果表明布鲁氏菌内存在一定数量的sRNA，而且sRNA在布鲁氏菌抵抗与适应巨噬细胞内恶劣环境中发挥重要作用。
本研究通过对布鲁氏菌在营养胁迫条件下的转录组测序、差异基因表达分析，对布鲁氏菌在营养胁迫条件下的毒力、致病、代谢等分子机制有了一定的认识。结合转录组测序和生物信息学预测对布鲁氏菌的全基因组进行扫描分析，寻找布鲁氏菌的sRNA，并对sRNA在布鲁氏菌适应环境压力和胞内生存中发挥的作用及其调控的靶基因进行了详细的探讨，为研究布鲁氏菌抵抗环境压力及胞内生存机制提供了重要线索。
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.
引文
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