流产布鲁氏菌毒力基因筛选鉴定及其功能研究
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摘要
布鲁氏菌病是一种重要的人畜共患病，其病原布鲁氏菌是一种兼性胞内寄生的革兰氏阴性菌，感染后，主要引起动物流产和人的波状热。布鲁氏菌无经典的毒力因子，如质粒，外毒素，溶细胞素，荚膜或内毒素特性的脂多糖分子，其毒力主要体现在入侵宿主细胞并在胞内存活的能力，基于此，布鲁氏菌能感染宿主建立慢性感染，较难被清除，因此研究布鲁氏菌建立慢性感染和胞内存活所需的基因对于阐明布鲁氏菌的致病机制具有重要意义。本研究主要采用信号标签转座子随机突变技术和基因芯片技术分别筛选鉴定布鲁氏菌建立慢性感染和胞内存活所需基因，为研究布鲁氏菌致病机制提供理论研究资料。
本研究中，我们通过逐步施压的方法，诱导流产布鲁氏菌S2308产生萘啶酸抗性（Nal）作为筛选标记，以抗性菌株为亲本，构建含8个信号标签的转座子随机突变库，共含有突变株3759株，以BABL/c小鼠为动物模型，共筛选2048株突变株，获得89株减毒株。通过步移PCR鉴定了24株减毒株的插入失活基因，其中获得一株粗糙型布鲁氏菌，鉴定为rfbE基因失活，rfbE基因为布鲁氏菌O-抗原转运系统ATP结合蛋白，为进一步研究该基因的功能，我们通过定向缺失技术构建流产布鲁氏菌rfbE基因缺失株，命名为rfbE，经结晶紫染色和热凝集试验确定rfbE为粗糙表型，生长曲线测定发现rfbE在平台期以后生长略慢于野生株，细胞感染试验发现rfbE缺失株提高了入侵巨噬细胞的能力，但明显减弱其胞内存活的能力，通过形态学分析和荧光染色发现rfbE诱导细胞肿胀坏死。通过对BABL/c小鼠模型致病性分析发现rfbE缺失株明显出现减毒现象，不能在小鼠模型上建立慢性感染。此研究表明，rfbE基因参与LPS合成，是布鲁氏菌毒力所必需的。
为深入研究rfbE缺失株的特性，我们通过基因芯片筛选rfbE缺失株差异转录基因，发现asp24基因在rfbE中出现明显的上调转录，随后，通过荧光定量PCR和免疫印迹试验表明Asp24上调表达不是粗糙型布鲁氏菌共有的特征，而是与O-抗原ABC转运子缺失引起O-抗原胞内聚集相关，发现一种新的Asp24上调机制。荧光染色试验表明Asp24不是粗糙型布鲁氏菌致死巨噬细胞的原因，酸诱导性试验表明Asp24确实能在酸性条件下被诱导表达，然而Asp24并不是布鲁氏菌抵抗酸性环境所必需的组分。进一步研究发现，Asp24蛋白在胞内环境下被精确调控表达对于布鲁氏菌胞内存活来说是必不可少的。
布鲁氏菌能在宿主体内建立慢性感染与其胞内存活的特性是分不开的，因此鉴定布鲁氏菌胞内存活所需基因对于理解其胞内存活机制非常重要，后续试验中我们通过基因芯片技术筛选鉴定了布鲁氏菌在巨噬细胞内存活状态下差异转录基因，发现7.82%的基因上调转录，5.4%的基因下调转录，荧光定量PCR验证芯片差异大于5倍的基因，共发现14明显差异转录基因，分别为：AraC转录调控子（AraC），DnaA结构域蛋白（Ddp），雌烯醇转录调控子（EryD），碱性磷酸酶（Alp），鞭毛基因棒状蛋白（FlgF），四型分泌系统VirB9组分（VirB9），原儿茶酸2,3双加氧酶（Hpcd），醛脱氢酶家族蛋白（ALDH），主要推动家族转运子（MFS），镍离子转运透化酶和四个假定蛋白基因（BruAb1_1814，BruAb1_0475，BruAb1_1926，BruAb1_0292），分别参与执行各种不同的功能，本研究表明，布鲁氏菌通过调控多种基因的表达来适应胞内存活的条件，是一个复杂的调控过程。
流产布鲁氏菌胞内诱导ddp基因，编码一个DNA结合域蛋白，该基因缺失株表现粗糙型布鲁氏菌的特征，但不能被结晶紫染色，免疫印迹试验分析发现， ddp缺失株仅能合成部分O抗原。粘附入侵试验发现ddp缺失株明显提高了入侵巨噬细胞和上皮样细胞的能力，约为S2308株的10-20倍，另外， ddp缺失株与粗糙型布鲁氏菌相似也能诱导巨噬细胞死亡，减弱在巨噬细胞内的复制能力，与S2308相比，明显激活巨噬细胞辅助抗原递呈分子CD40、CD80、CD86和细胞因子IL-6，IL-10和TNF-α的转录。动物试验显示， ddp缺失株不能在小鼠体内建立慢性感染，减弱对小鼠的致病力。本研究表明ddp基因是布鲁氏菌重要的致病基因，是布鲁氏菌毒力所必需的。
总之，本论文通过信号标签随机突变技术和基因芯片技术筛选鉴定了布鲁氏菌建立慢性感染和胞内存活时所需要的基因，研究了布鲁氏菌rfbE基因的功能，发现了一种新的Asp24上调机制，探讨布鲁氏菌胞内存活的诱导基因，研究了胞内诱导ddp基因的功能，为布鲁氏菌致病机制研究提供了理论研究资料。
Brucellosis is an important zoonotic disease worldwide, and the causative agent is Brucella spp.,which is a species of facultative, intracellular, Gram-negative bacteria that induces abortion and causessterility in domesticated mammals and chronic undulant fever in humans. Brucella has no classicalvirulence factors including plasmid, exotoxins, cytolysins, capsules or endotoxic lipopolysaccharide(LPS) molecules. Virulence of the Brucella species depends on their survival and replication in hostcells, based on this property, Brucella can establish chronic infection in host and it is difficult to beeliminated in vivo, so it is great of significance to identify genes necessary for chronic infection andintracellular survival in Brucella. In this study, signature-tagged mutagenesis (STM) and DNAmicroarray was used to identify the related genes in chronic infection and intracellular survival,respectively, which provide theoretical research materials for Brucella pathogenesis.
In this study, Brucella abortus strain S2308was induced to resist nalidixic acid (Nal) by graduallyincreasing antibiotic concentration, random mutant libraries with8different tags were constructed usingthe Nal resistance strain as a receptor, including a total of3,759mutants. Among these,2,048mutantswere screened in BABL/c mouse model and89mutants were attenuated for mouse. Inactivated genes of24attenuated mutants were identified by genome waking PCR, of these, one mutant showed roughphenotype with disrupted rfbE gene which encodes O-antigen export system ATP-binding protein. Tofurther study the function of rfbE gene, B. abortus rfbE gene deletion strain was constructed byhomologous recombination, designated as rfbE strain, which rough phenotype was confirmed by violetstaining and heat agglutination. The growth curve showed that rfbE strain has a slightly weaker growththan wild-type strain after the stationary phase. Cell infection assay revealed that rfbE strain enhancedthe ability to invade into macrophages, but reduced intracellular survival, furthermore, rfbE straininduce macrophage oncosis and necrosis by morphological observation and fluorescent staining. Mouseinfection assay showed that rfbE strain cannot establish chronic infection in BABL/c mouse. Theseresults indicated that rfbE gene is involved in LPS synthesis and is necessary for Brucella virulence tomouse.
To further study the properties of rfbE mutant strain, DNA microarray was used to indentifydifferentially transcripts between rfbE and wild-type (wt) strain, showing that asp24gene issignificantly upregulated in rfbE mutant, qRT-PCR and western blotting confirmed that the Asp24upregulation is not associated with rough phenotype, but associated with O-antigen intracellularsynthesis due to disruption of O-antigen export system, indicating that a novel mechanism of Asp24regulation is found. However, fluorescent staining showed that Asp24upregulation is not the cause ofrough Brucella-induced macrophage death, and although Asp24is induced in acid condition, it is notnecessary for Brucella to resist acidified environment. In the further study, we confirmed that Asp24accurately regulation in intracellular environment plays an indispensable role in survival for Brucella.
Brucella establishing chronic infection in host is associated with the ability to intracellular survival, so identifying genes necessary for intracellular survival may hold the key to understanding Brucellainfection. In the present study, a microarray assay was used to identify genes differentially expressed inBrucella within RAW264.7cells. The results show that7.82%of Brucella genes were up-regulated and5.40%were down-regulated. qRT-PCR analysis further verified that the levels of14Brucella geneswere up-regulated more than5-fold, including AraC transcriptional regulator (AraC), DnaA domainprotein (Ddp), erythritol transcriptional regulator,(EryD) alkaline phosphatase (Alp), flagellar basalbody rod protein (FlgF), and type IV secretion system protein VirB9(VirB9), homoprotocatechuate2,3-dioxygenase (Hpcd), aldehyde dehydrogenase family of proteins (ALDH), major facilitator familytransporters (MFS), nickel transporter permease (NikC), and four hypothetical proteins, encoded byBruAb1_1814, BruAb1_0475, BruAb1_1926, and BruAb1_0292, which14genes play different roles inbiological pathway. These results revealed that Brucella adapts to intracellular environment under thecontrol of extensive genes regulation, which is a complex process.
The ddp gene is induced expression in intracellular environment which encode a DNA bindingdomain, B. abortus ddp gene deletion mutant shows the characteristics of rough-type strain, but cannotbe stained purple by crystal violet. Western blotting showed that ddp strain reduced O-antigensynthesis compared to wt strain. Adherence and invasion assay showed that ddp strain enhanced theability to adhere to and invade into macrophages and epithelioid cells, thus increased of>10-20fold.Moreover, as same as the rough-type strains, ddp strain can also induce macrophage death and reducedintracellular survival within macrophages. Compared to S2308strain, ddp strain induced upregulationof antigen presenting related molecules CD40, CD80, CD86and pro-inflammatory cytokines IL-6，IL-10and TNF-α. Additionally, ddp strain cannot establish chronic infection in BABL/c mouse. Allthe results indicated that ddp gene is a virulent-related factor and it is necessary for Brucella to establishchronic infection.
In summary, the paper identifies Brucella genes necessary for establishing chronic infection andintracellular survival using the technologies of STM and DNA microarray. The works included studyingthe function of rfbE gene, discovering a novel mechanism of Asp24upregulation, identifying genesinduced in intracellular environment and clarifying the function of ddp gene, which provide the novelinformation for Brucella pathogenesis research.
本研究中，我们通过逐步施压的方法，诱导流产布鲁氏菌S2308产生萘啶酸抗性（Nal）作为筛选标记，以抗性菌株为亲本，构建含8个信号标签的转座子随机突变库，共含有突变株3759株，以BABL/c小鼠为动物模型，共筛选2048株突变株，获得89株减毒株。通过步移PCR鉴定了24株减毒株的插入失活基因，其中获得一株粗糙型布鲁氏菌，鉴定为rfbE基因失活，rfbE基因为布鲁氏菌O-抗原转运系统ATP结合蛋白，为进一步研究该基因的功能，我们通过定向缺失技术构建流产布鲁氏菌rfbE基因缺失株，命名为rfbE，经结晶紫染色和热凝集试验确定rfbE为粗糙表型，生长曲线测定发现rfbE在平台期以后生长略慢于野生株，细胞感染试验发现rfbE缺失株提高了入侵巨噬细胞的能力，但明显减弱其胞内存活的能力，通过形态学分析和荧光染色发现rfbE诱导细胞肿胀坏死。通过对BABL/c小鼠模型致病性分析发现rfbE缺失株明显出现减毒现象，不能在小鼠模型上建立慢性感染。此研究表明，rfbE基因参与LPS合成，是布鲁氏菌毒力所必需的。
为深入研究rfbE缺失株的特性，我们通过基因芯片筛选rfbE缺失株差异转录基因，发现asp24基因在rfbE中出现明显的上调转录，随后，通过荧光定量PCR和免疫印迹试验表明Asp24上调表达不是粗糙型布鲁氏菌共有的特征，而是与O-抗原ABC转运子缺失引起O-抗原胞内聚集相关，发现一种新的Asp24上调机制。荧光染色试验表明Asp24不是粗糙型布鲁氏菌致死巨噬细胞的原因，酸诱导性试验表明Asp24确实能在酸性条件下被诱导表达，然而Asp24并不是布鲁氏菌抵抗酸性环境所必需的组分。进一步研究发现，Asp24蛋白在胞内环境下被精确调控表达对于布鲁氏菌胞内存活来说是必不可少的。
布鲁氏菌能在宿主体内建立慢性感染与其胞内存活的特性是分不开的，因此鉴定布鲁氏菌胞内存活所需基因对于理解其胞内存活机制非常重要，后续试验中我们通过基因芯片技术筛选鉴定了布鲁氏菌在巨噬细胞内存活状态下差异转录基因，发现7.82%的基因上调转录，5.4%的基因下调转录，荧光定量PCR验证芯片差异大于5倍的基因，共发现14明显差异转录基因，分别为：AraC转录调控子（AraC），DnaA结构域蛋白（Ddp），雌烯醇转录调控子（EryD），碱性磷酸酶（Alp），鞭毛基因棒状蛋白（FlgF），四型分泌系统VirB9组分（VirB9），原儿茶酸2,3双加氧酶（Hpcd），醛脱氢酶家族蛋白（ALDH），主要推动家族转运子（MFS），镍离子转运透化酶和四个假定蛋白基因（BruAb1_1814，BruAb1_0475，BruAb1_1926，BruAb1_0292），分别参与执行各种不同的功能，本研究表明，布鲁氏菌通过调控多种基因的表达来适应胞内存活的条件，是一个复杂的调控过程。
流产布鲁氏菌胞内诱导ddp基因，编码一个DNA结合域蛋白，该基因缺失株表现粗糙型布鲁氏菌的特征，但不能被结晶紫染色，免疫印迹试验分析发现， ddp缺失株仅能合成部分O抗原。粘附入侵试验发现ddp缺失株明显提高了入侵巨噬细胞和上皮样细胞的能力，约为S2308株的10-20倍，另外， ddp缺失株与粗糙型布鲁氏菌相似也能诱导巨噬细胞死亡，减弱在巨噬细胞内的复制能力，与S2308相比，明显激活巨噬细胞辅助抗原递呈分子CD40、CD80、CD86和细胞因子IL-6，IL-10和TNF-α的转录。动物试验显示， ddp缺失株不能在小鼠体内建立慢性感染，减弱对小鼠的致病力。本研究表明ddp基因是布鲁氏菌重要的致病基因，是布鲁氏菌毒力所必需的。
总之，本论文通过信号标签随机突变技术和基因芯片技术筛选鉴定了布鲁氏菌建立慢性感染和胞内存活时所需要的基因，研究了布鲁氏菌rfbE基因的功能，发现了一种新的Asp24上调机制，探讨布鲁氏菌胞内存活的诱导基因，研究了胞内诱导ddp基因的功能，为布鲁氏菌致病机制研究提供了理论研究资料。
Brucellosis is an important zoonotic disease worldwide, and the causative agent is Brucella spp.,which is a species of facultative, intracellular, Gram-negative bacteria that induces abortion and causessterility in domesticated mammals and chronic undulant fever in humans. Brucella has no classicalvirulence factors including plasmid, exotoxins, cytolysins, capsules or endotoxic lipopolysaccharide(LPS) molecules. Virulence of the Brucella species depends on their survival and replication in hostcells, based on this property, Brucella can establish chronic infection in host and it is difficult to beeliminated in vivo, so it is great of significance to identify genes necessary for chronic infection andintracellular survival in Brucella. In this study, signature-tagged mutagenesis (STM) and DNAmicroarray was used to identify the related genes in chronic infection and intracellular survival,respectively, which provide theoretical research materials for Brucella pathogenesis.
In this study, Brucella abortus strain S2308was induced to resist nalidixic acid (Nal) by graduallyincreasing antibiotic concentration, random mutant libraries with8different tags were constructed usingthe Nal resistance strain as a receptor, including a total of3,759mutants. Among these,2,048mutantswere screened in BABL/c mouse model and89mutants were attenuated for mouse. Inactivated genes of24attenuated mutants were identified by genome waking PCR, of these, one mutant showed roughphenotype with disrupted rfbE gene which encodes O-antigen export system ATP-binding protein. Tofurther study the function of rfbE gene, B. abortus rfbE gene deletion strain was constructed byhomologous recombination, designated as rfbE strain, which rough phenotype was confirmed by violetstaining and heat agglutination. The growth curve showed that rfbE strain has a slightly weaker growththan wild-type strain after the stationary phase. Cell infection assay revealed that rfbE strain enhancedthe ability to invade into macrophages, but reduced intracellular survival, furthermore, rfbE straininduce macrophage oncosis and necrosis by morphological observation and fluorescent staining. Mouseinfection assay showed that rfbE strain cannot establish chronic infection in BABL/c mouse. Theseresults indicated that rfbE gene is involved in LPS synthesis and is necessary for Brucella virulence tomouse.
To further study the properties of rfbE mutant strain, DNA microarray was used to indentifydifferentially transcripts between rfbE and wild-type (wt) strain, showing that asp24gene issignificantly upregulated in rfbE mutant, qRT-PCR and western blotting confirmed that the Asp24upregulation is not associated with rough phenotype, but associated with O-antigen intracellularsynthesis due to disruption of O-antigen export system, indicating that a novel mechanism of Asp24regulation is found. However, fluorescent staining showed that Asp24upregulation is not the cause ofrough Brucella-induced macrophage death, and although Asp24is induced in acid condition, it is notnecessary for Brucella to resist acidified environment. In the further study, we confirmed that Asp24accurately regulation in intracellular environment plays an indispensable role in survival for Brucella.
Brucella establishing chronic infection in host is associated with the ability to intracellular survival, so identifying genes necessary for intracellular survival may hold the key to understanding Brucellainfection. In the present study, a microarray assay was used to identify genes differentially expressed inBrucella within RAW264.7cells. The results show that7.82%of Brucella genes were up-regulated and5.40%were down-regulated. qRT-PCR analysis further verified that the levels of14Brucella geneswere up-regulated more than5-fold, including AraC transcriptional regulator (AraC), DnaA domainprotein (Ddp), erythritol transcriptional regulator,(EryD) alkaline phosphatase (Alp), flagellar basalbody rod protein (FlgF), and type IV secretion system protein VirB9(VirB9), homoprotocatechuate2,3-dioxygenase (Hpcd), aldehyde dehydrogenase family of proteins (ALDH), major facilitator familytransporters (MFS), nickel transporter permease (NikC), and four hypothetical proteins, encoded byBruAb1_1814, BruAb1_0475, BruAb1_1926, and BruAb1_0292, which14genes play different roles inbiological pathway. These results revealed that Brucella adapts to intracellular environment under thecontrol of extensive genes regulation, which is a complex process.
The ddp gene is induced expression in intracellular environment which encode a DNA bindingdomain, B. abortus ddp gene deletion mutant shows the characteristics of rough-type strain, but cannotbe stained purple by crystal violet. Western blotting showed that ddp strain reduced O-antigensynthesis compared to wt strain. Adherence and invasion assay showed that ddp strain enhanced theability to adhere to and invade into macrophages and epithelioid cells, thus increased of>10-20fold.Moreover, as same as the rough-type strains, ddp strain can also induce macrophage death and reducedintracellular survival within macrophages. Compared to S2308strain, ddp strain induced upregulationof antigen presenting related molecules CD40, CD80, CD86and pro-inflammatory cytokines IL-6，IL-10and TNF-α. Additionally, ddp strain cannot establish chronic infection in BABL/c mouse. Allthe results indicated that ddp gene is a virulent-related factor and it is necessary for Brucella to establishchronic infection.
In summary, the paper identifies Brucella genes necessary for establishing chronic infection andintracellular survival using the technologies of STM and DNA microarray. The works included studyingthe function of rfbE gene, discovering a novel mechanism of Asp24upregulation, identifying genesinduced in intracellular environment and clarifying the function of ddp gene, which provide the novelinformation for Brucella pathogenesis research.
引文
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