【作者】 龚明玉；

【导师】 黄新祥；

【作者基本信息】 江苏大学 ， 临床检验诊断学， 2014， 博士

【摘要】 背景伤寒沙门菌(Salmonella enterica serovar Typhi,S. Typhi)作为一种重要的人类肠道致病菌,从外界环境进入宿主的过程中,系统基因表达调节十分重要。近年来生物信息学、DNA芯片技术、高通量测序技术的发展和应用,使得生物系统基因表达研究进展迅速。在各种生物体内除蛋白质外还存在许多非编码RNA(non-coding RNA, ncRNA)具有调节基因表达作用。这些ncRNA大多在转录后水平调节靶基因表达,参与细菌的各种生理反应,包括代谢调节、群体感应、毒力、铁代谢、氧化应激等。ncRNA的鉴定、功能和调节机制研究已成为当今生物医学的研究热点。目的本研究小组前期通过高通量测序结合应用生物信息学分析,在S. Typhi中发现了许多新的ncRNAs表达迹象。本研究旨在对S. Typhi中一种新的ncRNA (malS-5’UTR)的分子特征进行鉴定,并对其表达特性、对基因表达影响和作用机制开展深入研究。方法1. malS-5’UTR的分子特征鉴定利用高通量测序获得的信息,设计特异性的引物和探针,通过RT-PCR和Northern blot,对malS-5’UTR在S. Typhi中的表达进行验证,利用RACE实验分析malS-5’UTR的转录起始点和终止点。2. malS-5’UTR在不同生长条件下的表达特性分析以Northern blot和qRT-PCR分析S. Typhi在不同生长时期malS-5’UTR的表达特点。应用同样手段分析5. Typhi在不同应激条件(包括酸、氧及高渗环境下)malS-5’UTR的表达特性,并分析各种应激条件下σ因子对malS-5’UTR的表达调控作用。3.基因缺陷株的表型分析：(1)在不同条件下生长曲线的测定以生长时间为横坐标,OD600值为纵坐标绘制生长曲线,观察S. Typhi的malS-5’UTR基因缺陷株和对照株在普通条件和酸、氧及高渗应激条件下的生长状况。(2)动力实验测定将S. Typhi的malS-5’UTR基因缺陷株、bax基因缺陷株和对照株接种于0.3%琼脂糖动力平板,37-C培养一定时间后测量菌圈直径,以分析细菌动力。(3)对HeLa细胞的侵袭实验将malS-5’UTR基因缺陷株、bax基因缺陷株和对照株在等渗LB中培养至对数期,按MOI20：1加入HeLa细胞培养孔,培养90min后,将部分细胞破膜,收集菌体后涂板,过夜培养并计菌落数代表细菌粘附细胞水平(T0)；另将部分细胞加入庆大霉素继续培养90min后,按上述方法操作,菌落数代表细菌侵袭水平(T90),以T90/T0代表细菌对HeLa细胞的侵袭能力。4. malS-5’UTR和box对S. Typhi系统基因表达影响分析采用基因组芯片分析技术,分析S. Typhi系统基因转录水平表达。将S. Typhi的malS-5’UTR基因缺陷株、bax基因缺陷株和对照株在等渗LB中培养4h,提取总RNA,反转录并荧光标记cDNA,与基因组芯片杂交、扫描,将荧光信号数据化并比较菌株间的系统基因表达谱差异。选择部分基因,用实时荧光定量PCR分析其在菌株间的表达差异,以验证芯片分析结果。5. malS-5’UTR对其靶基因bax表达影响分析构建malS-5’UTR表达重组质粒pBAD-malS-5’UTR,并电转至S. Typhi野生株,用阿拉伯糖诱导malS-5’UTR高表达,用Northern blot和qRT-PCR进行分析bax的mRNA水平。采用了自杀质粒介导的同源重组方法,在S. Typhi野生株染色体bax基因加3X Flag标签序列,然后将pBAD-malS-5’UTR导入该菌株,用阿拉伯糖诱导malS-5’UTR高表达,用Western blot观察Bax蛋白水平的变化。6. malS-5’UTR影响S. Typhi侵袭力作用机制分析将pBAD-malS-5’UTR及空质粒pBAD分别电击转化入S. Typhi的bax基因缺陷株中,用qRT-PCR检测侵袭相关基因invF的表达水平,并观察不同菌株对HeLa细胞侵袭能力,以分析malS-5’UTR对5. Typhi侵袭力的影响是否与其靶基因bax相关。结果1. malS-5’UTR分子特征鉴定通过5. Typhi转录组分析发现了一个236个核苷酸由bax的负链基因所编码的转录产物,(从bax基因起始密码子上游99nt到137nt),经Northern blot和RT-PCR分析,确定其在S. Typhi中有表达,且全长约有2000nt。5’-RACE实验结果显示其转录起始点位于malS基因起始密码子的上游453nt处。3’-RACE实验有多个结果,最长的一个片段位于malS基因起始密码子的下游1112nt。考虑到在Northern分析中并没有发现多个不同的片段,malS-5’UTR和malS重叠且和malS转录方向相同,结合生物信息学基因启动子分析结果,认为与bax的mRNA互补的转录产物实际就是malS的mRNA的5’非翻译区(5’untranslated region,5’UTR),因此命名其为malS-5’UTR。2. malS-5’VTR在S. Typhi中的表达特性Northern blot和qRT-PCR结果表明,malS-5’UTR在S. Typhi各生长时期和应激条件下均有表达,且在迟滞期(OD600=0.4)表达量最高,在酸、氧及高渗应激下的表达量均下降。在各种应激条件下,rpoE和rpoS缺陷株中malS-5’VTR的表达均少于野生株,表明其转录与RpoE和RpoS相关。3. malS-5’UTR基因缺陷株的表型分析(1)对伤寒沙门菌生长的影响与野生株相比,malS-5’UTR基因缺陷株的生长速度明显减慢,表明malS-5’UTR可以促进细菌生长；在酸及高渗应激时,malS-5’UTR缺陷株的生长无明显差异；在氧应激时,malS-5’UTR基因缺陷株的生长速度明显减慢,表明malS-5’UTR可以促进细菌在氧应激下的生长。(2)对伤寒沙门菌动力的影响动力分析表明,与野生株相比,malS-5’UTR缺陷株的动力圈明显增大,表明malS-5’UTR参与了沙门菌动力的调节。(3)对伤寒沙门菌侵袭力的影响与野生株相比,malS-5’UTR基因缺陷株对上皮细胞的侵袭力增强,表明malS-5’UTR可能参与了细菌侵袭力的调节。4. malS-5’UTR对S. Typhi系统基因表达影响分析芯片分析结果显示,与野生株相比,malS-5’UTR缺陷株中有334个基因表达发生明显变化,其中254个基因表达上调,主要包括鞭毛、动力和侵袭相关基因,有80个基因表达下调,主要为一些核糖体和代谢相关的基因；为了验证芯片结果的可靠性,选取其中部分表达变化的基因进行荧光定量PCR验证,结果显示所选基因表达变化与基因芯片分析结果一致。研究表明malS-5’UTR参与S. Typhi系统基因表达调节。5. malS-5’UTR对其靶基因bax表达影响分析Northern blot和qRT-PCR分析结果显示,随着malS-5’UTR表达诱导时间的延长,bax的mRNA水平逐渐减少,表明malS-5’UTR可降低bax mRNA的稳定性。Western blot分析结果显示,和空载体对照株比较,随着诱导时间的延长,Bax蛋白水平逐步下降。这些研究结果表明,Bax的表达受到malS-5’UTR的负向调节。6. malS-5’UTR影响S. Typhi侵袭力作用机制分析(1)Bax对S. Typhi侵袭力影响分析与野生株相比,bax基因缺陷株对上皮细胞的侵袭力增强,表明Bax可能参与了细菌侵袭力的调节。基因组芯片分析结果显示,与野生株相比,bax缺陷株中有67个基因表达发生明显变化,其中55个基因表达上调,主要为SPI-1、侵袭和毒力相关基因；有12个基因表达下调,主要为代谢相关基因。用qRT-PCR分析其中部分基因的表达,结果显示与基因芯片分析结果一致。研究表明Bax可能是S. Typhi侵袭的负调节因子,malS-5’UTR可通过抑制Bax的表达而增加S Typhi的侵袭力。(2) malS-5’UTR影响S. Typhi侵袭力与Bax相关性分析和含有空载体的bax基因缺陷株比较,在bax基因缺陷株中malS-5’UTR高表达后,侵袭调节基因invF的mRNA水平显著增加,同时细菌对HeLa细胞的侵袭力也明显增强。结果表明malS-5’UTR对侵袭的影响并不完全通过Bax,也可能通过其它途径进行。结论1.首次鉴定了S. Typhi中存在表达的malS基因上游一段非编码区为malS-5’UTR。2.malS-5’UTR基因缺失可增强细菌的动力和对HeLa细胞的侵袭力。3.malS-5’UTR基因缺失后能导致细菌发生系统性基因表达变化,涉及鞭毛结构和功能、侵袭、调节蛋白及部分代谢相关的基因。4.malS-5’UTR高表达降低靶基因bax的表达。5.Bax可能是伤寒沙门菌侵袭相关的一个负调节因子。6.malS-5’UTR可通过抑制bax表达,增强沙门菌的侵袭力,但不是唯一途径。更多还原

【Abstract】 BackgroundSalmonella enterica serovar Typhi (S. Typhi) is an important human intestinal bacteria. The regulation of gene expression play an important role in the process of entry to the host from environment. In recent years, with the application of bioinformatics, DNA microarrays and high-throughput sequencing, research on the gene expression regulation of biological system has been developed rapidly. Besides proteins, a great number of the noncoding RNAs (ncRNAs) were found involving in the gene expressional regulation network in various organisms. Researchers have found that ncRNAs mainly regulate the expression of target genes at the posttranscriptional level. These target genes play crucial regulatory roles in various physiological responses in bacterial, including the regulation of metabolism, quorum sensing, response to oxidative stress, iron homeostasis and virulence regulation of pathogens. Nowadays, the identification, function and regulatory mechanism of ncRNAs has already been the focus in biomedical research.ObjectiveThe expression indication of many new ncRNAs was discovered in S. Typhi by high-throughput sequencing and bioinformatics analysis in our group. The purpose of this work was to identify the molecular characteristics of a new ncRNA, malS-5’UTR, in S. Typhi, and further investigate its expressional pattern, the effect on the gene expression regulation, mechanisms of action.Methods1. Identification of the molecular characteristics of malS-5’UTR:According to the information obtained from high-throughput sequencing, specific probe and primers were designed, and the expression of malS-5’UTR in S. Typhi was confirmed by RT-PCR and Northern hybridization.5’-and3’-rapid amplification of cDNA ends (RACE) analysis were used to find the transcription initiation site and transcription termination site of malS-5’UTR respectively. The results of Northern blot and RACE were used to determine the full length of malS-5’UTR.2. Analysis of malS-5’UTR expression characteristic under different growth conditions:Northern blot and qRT-PCR assays were used to investigate the expression levels under different growth phases. The same analysis was also used to investigate the expression pattern of malS-5’UTR under different environmental stimuli, namely, acidic, oxidative, and hyperosmotic stresses, and the influence of rpoE and rpoS on the expression of the malS-5’UTR.3. Phenotype analysis of gene deletion mutants(1) the assay of growth curve of S. Typhi under different conditions:The growth of wild type S. Typhi and malS-5’UTR deletion mutant strains were assessed. Strains were subjected to growth under normal as well as stress conditions and the absorbance of the culture media was taken hourly over a time period to determine the growth curve.(2) motility assays:Motility agar was used to characterize the motility phenotypes of malS-5’UTR deletion mutant, box deletion mutant and wild type strain of S.Typhi. These strains were inoculated onto semi-solid LB plates (0.3%agar) and incubated at37℃for several hours. The diameter of motility circle was measured in order to assess the motility of bacteria.(3) Invasion capacity assay with HeLa cells:malS-5’UTR deletion mutant, box deletion mutant and wild type strain were incubated in LB medium to OD600of0.4. HeLa cells were seeded into24-well plates and infected by bacteria as a MOI of20:1. After infection90min, they were then either lysed and incubated on LB plate, and the number of clone was regarded as the level of adherence (To), or incubated in medium containing gentamicin for a further90min to kill extracellular bacteria and then lysed to assess the level of invasion (T90). The value of T90/T0represents the bacterial capacity invading into HeLa cells.4. Gene expression profile analysis of S. Typhi regulated by malS-5’UTR and Bax:The genome-wide gene expression profile was analyzed by genomic microarray assay. malS-5’UTR deletion mutant, box deletion mutant and wild type strain were incubated in LB medium for4h. Total RNAs of mutant and wild type were extracted, transcribed into cDNAs and reciprocally labeled with cy3-and cy5-dCTP one time. After Hybridization with Salmonella genomic DNA microarray, the slides were scanned, and the signals were digitalized. The gene expression difference between the wild type strain and mutant strain was analyzed. To confirm the findings of the microarray experiments, some genes with expressional change in the microarray assay were selected for qRT-PCR analysis.5. Analysis of effect malS-5’UTR on the expression of the target bax gene:The recombinant plasmid pBAD-malS-5’UTR was constructed. The wild type strain was transformed with the recombinant plasmid pBAD-malS-5’UTR, which expressed a240nt transcript of the malS-5’UTR in the strain after induction with L-arabinose. After the overexpression of the malS-5’UTR, the level of bax mRNA was investigated with Northern blot and qRT-PCR. To further investigate the effect of the malS-5’UTR on the expression of Bax protein, a Bax-3xFLAG fusion strain was constructed by homologous recombinant and transformed with the expressional plasmid of malS-5’UTR (pBAD-5’UTR). L-Arabinose (0.2%w/v) was added to the cultures to induce the overexpression of the malS-5’UTR. The effect of malS-5’UTR overexpression on the expression of Bax protein was assessed by immunoblotting with anti-FLAG-tag antibody.6. Analysis of the mechanism of malS-5’UTR regulating invasiveness:To gain further insight into the mechanism of the malS-5’UTR in affecting bacterial invasion, pBAD-5’UTR and pBAD empty plasmid were transferred into the bax mutant of S. Typhi. The mRNA level of invasion-associated gene invF was measured with qRT-PCR, and bacterial invasiveness to HeLa cells was also examined.Results1. Identification of the molecular characteristics of malS-5’UTR:The analysis with RNAseq of S. Typhi showed a236-nt transcript encoded by the minus strand of bax, extending from137nt downstream to99nt upstream from the start codon of bax. The transcript was confirmed by Northern blot and RT-PCR assay and its length was about2000nt.5’-RACE analysis showed the transcription start site was located453bp upstream from the malS start codon.3’-RACE detected different ends of the transcript, and the longest located1112bp downstream from the start codon of malS. However, no relative fragments was seen in the result of Northern hybridization assay. Considering the same transcriptional direction of the transcript with the malS, combining the results of RACE, Northern blot, and bioinformatics analysis of the promoter, the transcript encoding region overlaping malS is the5’-untranslated region of the malS gene, and therefore named as matS-5’UTR.2. Expression characteristics of malS-5’UTR in S. Typhi:The expression characteristics of the malS-5’UTR were investigated with Northern blot and qRT-PCR. Results reflect the fact that the malS-5’UTR is expressed in all growth phases of S. Typhi, with an highest expression during the lag phase (OD6000.4). The expression of the malS-5’UTR in S. Typhi was decreased under various stresses. The relative expression levels of the malS-5’UTR decreased in the rpoE and rpoS mutant strains compared with the wild type strains under stresses. The result suggests that the transcription of malS-5’UTR is related with sigma factors RpoE and RpoS.3. Phenotype analysis of the malS-5’UTR mutant:(1) Growth assay:Under normal condition, the growth rate of the AmalS-5’UTR mutant was slightly lower than the wild type, which indicates that the malS-5’UTR can accelerate the growth of bacteria. The growth curves under acidic and high osmotic stress conditions showed that there is no significant difference between wild type strain and AmalS-5’UTR mutant. Under oxidative stress condition, the growth rate of the△malS-5’UTR mutant was significantly lower than that of the wild type, which shows that the malS-5’UTR can accelerate the growth of bacteria under oxidative stress condition.(2) Motility assay:The motility experiment displayed a significant increase in migration from the spot of inoculation in the mutant AmalS-5’UTR compared with the wild type. This result suggests that the malS-5’UTR is involved in the regulation of motility in S. Typhi.(3) Invasiveness assay:The invasive capacity with HeLa cells of the malS-5’UTR deletion mutant was stronger than that of wild type strain. It indicates that malS-5’UTR is participated in the regulation of bacteria invasiveness. 4. Effect of malS-5’UTR on gene expression profiling:A comparison of the global transcription profiles revealed that334genes were differentially expressed in the malS-5’UTR mutant and the wild-type strain. Of these genes,254were up-regulated in the malS-5’UTR mutant compared with the wild type, including genes involved in flagellum, motility and invasion.80were down-regulated in the malS-5’UTR mutant, including ribosome-associated and the metabolism-associated genes. To confirm the findings of the microarray experiments, some genes with expression change in the microarray assay were selected for qRT-PCR analysis. Results indicated that the expression levels of selective genes from qRT-PCR analysis were consistent with the levels from microarray assay. It shows that the malS-5’UTR is participated in the systemic gene expression regulation in S. Typhi.5. Effect of malS-5’UTR on the expression of target box gene:The box mRNA level decreased gradually with increasing induction time by Northern blot and qRT-PCR analysis. These results indicate that the malS-5’UTR can decrease the stability of box mRNA. Compared with the control strain containing empty plasmid, the box gene protein level reduced gradually with increasing induction time by Western blot analysis. Together, these results indicate that the expression of Bax is negatively affected by the malS-5’VTR.6. Analysis of the mechanism of the malS-5’UTR action on invasion of S. Typhi:(1) Effect of Bax on the invasiveness of S. Typhi:The invasive capacity to HeLa cells of the bax deletion mutant strain was stronger than that of the wild type strain. It indicates that bax may be participated in the regulation of bacteria invasiveness. A comparison of the global transcription profiles revealed that67genes were differentially expressed in the bax mutant and the wild-type strain. Of these genes,55were up-regulated in the bax mutant, including SPI-1, virulence and invasion genes.12were down-regulated in the bax mutant, involving in the metabolism-associated genes. To confirm the findings of the microarray experiments, some genes with expressional change in the microarray assay were selected for qRT-PCR analysis. Results of qRT-PCR analysis was consistent with the the results of microarray analysis. It shows that the Bax is a invasion negative factor, and the malS-5’UTR may increase invasive capacity of S. Typhi by inhibiting the expression of Bax.(2) malS-5’UTR regulating the invasiveness of S. Typhi associated with the Bax. After over-expressing the malS-5’UTR in the deletion mutant of bax, the mRNA level of invF and invasiveness to HeLa cells were increased compared with the control strain, the bax mutant containing an empty plasmid. These finding suggest that the effect of malS-5’UTR on invasion may be partially dependent on its target bax gene. malS-5’UTR may affect the expression of invasion-associated gene and invasiveness by other ways.Conclusions1. A non-coding RNA, malS-5’UTR expressed in S. Typhi was first identified.2. The deletion of the malS5’-UTR can increase the bacterial motility and the invasiveness to the HeLa cells.3. The deletion of the malS5’-UTR led to the change of the expression of system genes, including flagellar structure and function, invasion, regulatory-protein, and some metabolism-related genes.4. The overexpression of the malS5’-UTR can reduce the expression of bax.5. Bax may be a negative factor of invasion in S. Typhi.6. ma/S-5’UTR increases bacterial invasiveness by inhibiting the expression of Bax, which is not a exclusive pathway.更多还原