【作者】 孙芝兰；

【导师】 孔健；

【作者基本信息】 山东大学 ， 发酵工程， 2012， 博士

【摘要】 乳杆菌是人和动物肠道的正常菌群,具有维持肠道内菌群平衡,提高机体免疫力,促进营养物质吸收等生理功能。同时乳杆菌在生长过程中,能够产生乳酸、乙酸、过氧化氢、细菌素等抑菌物质,对肠道病原菌有抑制作用,因此,乳杆菌对机体具有重要的益生作用。益生菌的筛选标准包括黏附和定植能力、无致病性、对病原菌有拮抗作用、不携带可转移的抗生素基因等。其中,对消化道表面的黏附和定植能力是重要的筛选指标,其黏附能力决定于细胞表层蛋白的组成及疏水特性。另外,益生菌对病原菌具有抑制作用,作用机制包括竞争有限的资源限制病原菌的生长、竞争病原菌黏附位点、分泌抗菌物质及失活毒素等。因此临床上应用益生菌剂来预防或治疗因抗药性病原菌引起的肠道感染及腹泻等疾病。但是目前大部分的研究只停留在作用效果上,缺乏对益生菌作用机理的研究。为了深入研究益生菌的作用机理,筛选功能明确、安全性高的益生菌,本文主要围绕高黏附乳杆菌的筛选、乳杆菌对病原菌黏附上皮细胞的抑制作用、黏附蛋白的结构和功能鉴定、乳杆菌抗生素抗性评价、乳杆菌表达载体构建五个方面展开研究,其结果如下：1.高黏附乳杆菌的筛选及益生特性的体外评价目前乳酸菌的益生作用已被人们所接受。在畜禽养殖业,益生菌剂替代抗生素以促进动物的生长已得到认可,但目前仍存在菌株单一、特异性不强等问题,因此优良益生菌株的选育是开发益生菌制剂的关键。本文将分离自健康鸡肠道的42株乳杆菌与结肠癌细胞系HT-29共同温浴,结果6株乳杆菌具有较高的黏附能力,经16S rDNA基因序列分析,分别鉴定为Lactobacillus crispatus K313、Lactobacillus crispatus K243、Lactobacillus helveticus2020T、Lactobacillus johnsonnii H31、Lactobacillus salivarius Z4、Lactobacillus salivarius K233。其中,Lb. crispatus K313黏附能力最强,黏附量为73.2bacteria/cell。随后将这6株乳杆菌与黏蛋白(mucin)、胞外基质蛋白Ⅰ型胶原(CnⅠ)、Ⅳ型胶原(CnIV)和纤连蛋白(FN)共同温育,结果菌株Lb. crispatus K243和K313表现出较高的黏附能力,其中Lb. crispatus K243与CnIV结合能力最高,结合量为1239bacteria/field。菌株Lb. crispatus K243和K313对酸和胆盐表现出较强的耐受力,在pH3.5的酸性环境中仍能缓慢生长,在0.1%的牛胆盐中培养6h,活菌数基本不变。Lb. crispatus K313和K243还具有胆固醇降解能力,胆固醇脱出率分别为60.8%和51.4%。Lb. crispatus K313和K243对正十六烷、氯仿的亲和力高达90%,而对乙酸乙酯的结合能力较弱,说明它们都具有疏水性的、碱性的细胞表面。透射电镜观察菌株K243和K313的超微结构显示,这2株菌的细胞壁表面都存在S层蛋白,用LiCl提取菌株K243和K313的S层蛋白,SDS-PAGE分析显示其大小分别为45kDa和60kDa,将其命名为SlpA和SlpB。用LiCl、蛋白酶K、胃蛋白酶、胰蛋白酶处理菌体细胞,SDS-PAGE分析显示SlpB对胃蛋白酶敏感,对胰蛋白酶不敏感,而SlpA对胃蛋白酶和胰蛋白酶都不敏感。经过LiCl处理后,Lb. crispatus K243和K313对胶原蛋白和HT-29细胞的黏附能力都明显下降,并且纯化的S层蛋白也能阻碍Lb. crispatus K243和K313对CnⅣ和HT-29细胞的黏附,说明S层蛋白参与了黏附过程。用LiCl处理菌株后,Lb. crispatus的形态变为椭圆或不规则状,表明S层蛋白对维持细胞正常形态发挥重要作用。去除S层蛋白后,这2株菌的存活率及对人工胃肠液的耐受性降低,说明S层蛋白可以保护宿主菌免受不利环境的影响。通过以上特性的研究,说明这2株菌具有益生菌的优良特性,可作为益生菌展开后续工作的研究。2. Lb. crispatus调节病原菌感染HT-29细胞所引起的炎症反应益生菌能预防和治疗因病原菌感染造成的腹泻等疾病,其作用机制包括减少病原菌定植,调节肠道菌群平衡及促进宿主免疫反应。益生菌一般通过共絮凝、竞争排斥等抑制病原菌的定植,本文研究表明,Lb. crispatus K313与病原菌Salmonella, braenderup H9812和Escherichia coli ATCC25922的共絮凝率分别为57.7%和84.8%,而Lb. crispatus K243与它们的共絮凝率分别为28.7%和26.2%,明显低于Lb. crispatus K313。随后将菌株K313和K243与S. braenderup H9812和E.coli ATCC25922共同感染HT-29细胞,结果Lb. crispatus K243和K313都能降低S. braenderup H9812和E. coli ATCC25922对HT-29细胞的黏附能力,并且菌株K313比K243表现出更强的抑制能力,此结果与共絮凝作用相吻合。当S. braenderup H9812单独感染HT-29细胞时,促炎性细胞因子IL-8、CXCL1和CCL20的转录水平是上调的,而当Lb. crispatus与S. braenderup H9812共同感染时,K313和243下调了IL-8的转录水平,下调幅度分别为42%和37%。另外,K313还下调了CXCL1和CCL20的转录水平,下调幅度为63%和41%。ELISA分析进一步验证了IL-8的分泌水平,结果表明Lb. crispatus K243和K313都能抑制由S. braenderup H9812感染HT-29所诱导的IL-8的分泌,抑制量分别为32.8%和47.0%。上述实验结果说明,Lb. crispatus K243和K313具有消弱S。braenderup H9812感染上皮细胞产生炎症反应的能力,为益生菌在临床上的潜在应用提供了理论参考。3.Lb. crispatus K313S-layer蛋白相关功能域研究如上所述,S层蛋白介导Lb. crispatus K313与HT-29细胞和胶原蛋白的黏附。为进一步阐明黏附机制,采取2种方法克隆了Lb. crispatus K313的S层蛋白基因：首先根据已报道S层蛋白基因序列,设计引物扩增S层蛋白的保守区,并结合ligation-anchored PCR扩增保守区的上下游序列,经拼接后得到S层蛋白基因全长,将其命名为slpA。第二种方法提取表层蛋白,进行SDS-PAGE分析,分离SDS-PAGE上的S层蛋白,并进行N端测序,根据得到的氨基酸序列设计简并引物,结合ligation-anchored PCR和Tail PCR扩增上下游序列。拼接后进行Blast比对共得到两个相邻的S层蛋白基因,命名为slpB和slpC。利用Real TimePCR分析slpA、slpB和slpC的表达模式,结果显示在检测条件下slpA是沉默的,slpB的转录水平是slpC的170倍左右,因此SlpB是Lb. crispatus K313中优势表达的S层蛋白。序列同源性比对显示,SlpB与SlpC氨基酸序列与所报道的卷曲乳杆菌S层蛋白氨基酸序列同源性较低。在slpB启动子下游有一段长约190bp的非翻译区UTLS,利用DNA重组技术将UTLS去除造成下游蛋白产量下降一半,说明UTLS对S层蛋白的高效表达至关重要。将SlpB截成一系列大小不等的片段,利用ELISA技术分析不同片段对胞外基质蛋白CnⅠ、CnⅣ、FN的结合能力,结果显示rSlpB1-501与CnIV和Cnl有显著水平的结合,分别是BSA的5.8和3.8倍,而不能结合FN。肽段rSlpB1-359与rSlpB1-379与CnⅠ、CnⅣ都有显著水平的结合,这说明与胶原蛋白结合功能域位于SlpB的N端。rSlpB1-359与胶原蛋白的结合能力相对于rSlpB1-379明显降低,将360-364五个氨基酸‘’VTVNV"中疏水氨基酸突变为亲水氨基酸‘TTTNT",突变后的rSlpB1-379与胶原蛋白结合能力下降,说明这五个氨基酸对SlpB与胶原蛋白的结合能力至关重要。将截断的片段分别与GFP融合表达,利用荧光酶标仪鉴定不同片段与细胞壁的锚定功能。结果显示SlpB细胞壁锚定结构域位于C端。用10%TCA处理去除细胞壁中磷壁酸成分,SlpB与细胞壁的结合能力下降60%,因此SlpB的结合受体可能是磷壁酸。rSlpB323-501-GFP与不同乳杆菌细胞壁的结合能力不同,对于本身具有S层蛋白的乳杆菌,SlpB与其有较高的结合能力,并且LiCl预处理能明显提高菌株与rSlpB323-501-GFP的结合量,对不含有S层蛋白的菌株,无论经过LiCl处理与否,它们与rSlpB323-501-GFP的结合量都非常低。因此SlpB与乳杆菌细胞表面的结合能力取决于乳杆菌本身是否含有S层蛋白。4.卷曲乳杆菌抗药性评价除了营养功能和益生作用,益生菌不能对人体或动物有任何负面效应,这就需要对益生菌进行安全性评价。其中,抗生素抗性是重要的评价指标。抗生素抗性基因型分析表明,Lb. crispatus K313携带氯霉素抗性基因cat、红霉素抗性基因ermB和四环素抗性基因tetL、tetM,而菌株K243携带有tetL、tetM抗性基因。克隆Lb. crispatus K313中的tetL、tetM基因,系统进化树分析显示TetL与已报道的TetL同源性较低,以高的自展值(100%)形成独立的分支。而TetM与已报道的TetM同源性较高,以97%的自展值聚类在一起。为进一步分析抗性基因的可转移性,以Lb. crispatus K243和K313为供体菌,Enterococcus faecalis K9为受体菌,采用滤膜杂交法,在含有抗生素平板上均没有筛选到抗性菌株,说明这2个菌株携带的抗性基因均不能转移到Enterococcus faecalis K9中,初步推断Lb. crispatus K243和K313在肠道内抗性相对稳定。5.乳杆菌表达载体构建以上实验均是在体外评价Lb. crispatus K243和K313的益生作用,为了在体内研究其定植、生长繁殖的动态变化,拟进行菌株荧光标记,通过质粒载体携带荧光蛋白基因在益生菌内有效表达,实时监测益生菌在动物体内的生长状态。鉴于目前乳杆菌质粒载体的不完善,本文分离和测序了植物乳杆菌的隐秘型质粒pD403,结果表明,pD403质粒全长2791bp,GC含量37%,含有两个开放阅读框orfl和orf2。ORFl含有318个氨基酸经比对其为一复制起始蛋白RepA。ORF2含有137个氨基酸,与Ralstonia pickettii12D的转录调节蛋白TrmB有31%的同源性。功能鉴定显示ORF2(Tra)对质粒在宿主菌中的复制不起决定性作用,但是Tra能明显提高质粒载体的转化率。pD403复制起始位点同源性比对显示pD403可能是一个滚环复制型质粒,并且RepA的同源性比对结果表明RepA与滚环复制第三家族的复制起始蛋白同源性较高。将pD403的复制子、氯霉素抗性基因、大肠杆菌克隆载体pUC19的复制子依次连接构建大肠杆菌／乳杆菌穿梭克隆载体pCD4032。检测了pCD4032的宿主范围,发现pCD4032能成功转化Lb. casei、Lb. plantarum、Lb. fermentum和Lb. brevis,转化效率从1.3×102到7×104转化子/μg DNA。扩增Lb. delbrueckii subsp. bulgaricus ATCC11842的乳酸脱氢酶启动子和终止子序列,并插入到pCD4032中构建表达载体pCD4033。以绿色荧光蛋白基因(gfp)为报告基因,将其连接到载体pCD4033启动子下游,获得重组质粒pCD4033-gfp,然后重组质粒电转化乳杆菌感受态细胞。在荧光显微镜下,所有的重组菌株都能观察到绿色荧光,而只含有空质粒pCD4033的重组菌株没有检测到绿色荧光。在经过条件优化后,重组子于30℃,摇动培养情况下荧光值能达到最高。上述结果说明gfp在这几株宿主菌中能成功表达,表达载体pCD4033能够在乳杆菌中用于异源基因的克隆与表达,不幸的是,载体pCD4033没能成功转化Lb. crispatus K243和K313。更多还原

【Abstract】 Lactobacilli are dominant inhabitants of humans and animals. They can maintain a balanced intestinal flora, improve immunity and promote the absorption of the nutrients. Also Lactobacillus can produce lactic acid, acetic acid, hydrogen peroxide, bacteriocins and have an inhibitory effect on enteric pathogens. Therefore, Lactobacillus has an important role of prebiotics on human and animals. Probiotic screening criteria include the adhesion and colonization ability, antagonistic to pathogens and no transferable antibiotc resistance gene. Adhesion and colonization determined by the composition and hydrophobic characteristics of surface layer protens is an important screening index. Besides, probiotics have an inhibitory effect on pathogens. The mechanisms of action include the inhibition of pathogen growth by competition for nutritional sources and adhesion sites, secretion of antimicrobial substances and toxin inactivation. Consequently, Lactobacillus was used in clinical to prevent and treat gastrointestinal infections and antibiotic-associated diarrhea diseases. But most of the researches just study the effects of prebiotics and lack of the mechanism of action of Lactobacillus. For the development of probiotics with a clear feature and high security, we studied mainly on the screening of the high adhesive Lactobacillus, the inhibition of pathogens adhesion to epithelia cells, the structure and function of the adhesion proteins, antibiotic resistance evaluation of intestinal Lactobacillus and construction of expression vectors for Lactobacillus, the results were described as follows:1. The isolation and in vitro evaluation of high adhesive Lactobacillus strainsCurrently, there is an increasing interest in the use of probiotics as an alternative strategy to antimicrobial compounds. But there are still many problems such as single strains and poor specificity. Thus, the breedings of the excellent probiotic strains are the key to the development of probiotics. In this study, six Lactobacillus strains isolated from chicken intestinal tract had the adhsion ability to HT-29cells. They were Lactobacillus crispatus K313, Lactobacillus crispatus K243, Lactobacillus helveticus2020T, Lactobacillus johnsonnii H31, Lactobacillus salivarius Z4and Lactobacillus salivarius K233. Lb. crispatus K313exhibited the highest adhesion capacity to HT-29cells (73.2bacteria/cell). The six Lactobacillus strains adherences to mucin and individual proteins of the mammalian extracellular matrix were tested. the high attachment to collagen type IV (1239bacteria/field) were observed with the strain Lb. crispatus K243. Lb. crispatus K243and K313showed srong tolerance to acid and bile salts. The two strains also had a cholesterol degradation ability and the rate were60.8%and51.4%. Both of strains showed the very high percentages adhered to hexadecane demonstrated hydrophobic properties in the cell surface of Lb. crispatus. A strong affinity to chloroform and a low adherence to ethyl acetate indicated the basic characteristics in the two strain surfaces. SDS-PAGE analysis revealed the presence of the potential S-proteins Slp A and SlpB in Lb. crispatus K243and K313. SlpB of the strain K313was sensitive to pepsin, but not to trypsin, while SlpA was sensitive to neither pepsin nor trypsin. The treatment with LiCl greatly reduced the adhesiveness of the two Lb. crispatus strains compared to the untreated bacterial cells, subsequently, purified S-proteins also exhibited the inhibition of Lb. crispatus adhesion to collagens and HT-29cells, which suggested the S-layer proteins might be involved in the adhesion process. After LiCl treatment, Lb. crispatus K313and K243become oval or irregular shape. Consequently, the S-protein play an important role in maintaining the normal morphology of the cells. After removal of S-proteins, the viability and tolerance of the two Lb. crispatus strains to simulated gastric and small intestinal juice were reduced, indicating the protective role of S-proteins against the hostile environments. All the results showed that the two strains have the excellent characteristics and can be used as probiotics to lauch a follow-up work.2. Lb. crispatus regulate the inflammation caused by pathogen infection in HT-29cellsProbiotics can prevent and treat diarrhea and other diseases caused by pathogens infections. The mechanism of action include reducing pathogen colonization, regulating the balance of intestinal flora and promoting the host immune response. Probiotics can inhibite the colonization of pathogen via coaggregation and competitive exclusion. In this work, the coaggregation between Lb. crispatus K313and Salmonella. braenderup H9812or Escherichia coli ATCC was57.7%and84.8%, however; in Lb. crispatus K243, the coaggregation was28.7%and26.2%, lower than K313. Lb. crispatus K313and K243exhibited the strong inhibitive activity against S. braenderup H9812and E. coli ATCC25922adhesion to HT-29cells. Exposure of polarized HT-29cells to S. braenderup increased the transcription yields of pro-inflammatory factors (IL-8, CXCL1and CCL20). The transcription of IL-8were down-regulated by42%and37%in the case of coinfection with both Lb. crispatus strains and S. braenderup H9812. CXCL1and CCL20transcription levels were down-regulated by63%and41%when HT-29cells were preincubated with the Lb. crispatus K313before infection with S. braenderup H9812. The production of IL-8was detected by IL-8ELISA. Lb. crispatus K243and K313inhibited the IL-8secretion triggered by S. braenderup H9812by32.8%and47.0%, indicating that the two isolates could attenuate the pro-inflammatory signaling induced by S. braenderup H9812, and have the potential for protecting the host against S. braenderup infection.3. The functional domains of an S-protein from Lactobacillus crispatus K313It was previously shown that the S-proteins covering the cell surface of Lactobacillus crispatus K313were involved in the adherence of this strain to human intestinal cell line HT-29. To further elucidate the structures and functions of S-layers, three putative S-protein genes(slpA, slpB, and slpC) of Lb. crispatus K313were amplified with two strategies. First, the conserved primers were synthesized based on the conserved part of the S-protein gene of Lb. crispatus, and PCR amplification was performed with the genomic DNA of Lb. crispatus K313as the template. The upstream and downstream regions of the conserved part of the S-protein gene were amplified using the ligation-anchored PCR. The DNA products were then purified, sequenced, and spliced, respectively. Subsequently, the obtained gene was named slp A. Second, the oligonucleotide complementary to the N-terminal amino acid sequence of S-protein isolated was synthesized. For cloning the upstream and downstream region, the Tail-PCR and ligation-anchored PCR was performed for chromosome walking. After splicing, two adjacent S-protein genes were obtained, and named slpB and slpC. Quantitative real time PCR analysis reveals that slpA was silent under the tested conditions, whereas the transcription of slpB was170-fold higher than that of slpC. Therefore, slpB was predominantly expressed in Lb. crispatus K313. Genetic truncation of the untranslated leader sequence (UTLS) of slpB results in a reduction in protein production, indicating that the UTLS contributed to the efficient S-protein expression. We obtained a set of N-and C-terminally truncated recombinant SlpB proteins by constructing a series of recombinant vectors in Escherichia coli. Collagen type I, collagen type IV and human plasma fibronectin were chosen as the extracellular matrix proteins to investigate the binding ability of SlpB using ELISA techniques. rSlpB1-501exhibited significant levels of attachment to collagen types IV and I, with levels4.8-fold and2.8-fold higher than the background level seen with the immobilized control protein BSA, respectively. No binding was observed to the plasma fibronectin. The N-terminal truncated peptides rSlpB1-190, rSlpB1-322, and rSlpB1-341and the C-terminal truncated peptides rSlpB323-501, rSlpB342-501, and rSlpB3go-501only exhibited a background level of binding to collagen types I and IV. However, binding of rSlpB1-359and rSlpB1-379to both collagen types was observed, suggesting that SlpB domain involved in the adherence to collagens is located at the N-terminus. The derivative rSlpB1-359was reduced in collagen binding, revealing the importance of the20aa-peptide of SlpB between359and379. The mutated rSlpB1-379peptides VTVNV364TTTNT had a reduced binding ability to collagens, indicating that the five amino acids "VTVNV" are critical for the adhsion of SlpB to collagens. The binding of truncated SlpB-GFP fusing proteins to CWFs isolated were tested. The results showed that the cell wall binding region was mapped to the C-terminus. The treated CWFs with10%TCA reduced the binding ability of SlpB by60%, suggesting that teichoic acid may be acting as the receptor of SlpB. Moreover, the binding ability of the C-terminus was variable among the Lactobacillus species (S-layer and non-S-layer producing strains). SlpB had the high binding capacity to the S-layer containing Lactobacillus. Moreover, LiCl pretreatment increased the binding level of rSlpB323-501-GFP. However, a low binding level of the rSlpB323-501-GFP peptide was detected with the native or LiCl treated cell, which do not express an S-layer. Therefore, the different binding capacities of SlpB depended on whether these strains possessed S-layers.4. The antibiotic resistance evaluation of Lb. crispatusIn addition to nutritional function and prebiotic effect, probiotics can not have any negative effects on human or animal. It needs to evaluate the safety of probiotics. One of the main principle is that the evaluation of the antibiotic resistance pattern. Antibiotic resistance genotype analysis showed that Lb. crispatus K243and K313harbored tetracycline resistance gene tetL and tetM. Lb. crispatus K313also harbored chloramphenicol resistance gene cat and erythromycin resistance gene ermB. tetL and tetM were cloned from Lb. crispatus K313. Phylogenetic tree of homologs analysis indicated that TetL are different from all previously described TetL and form an independent branch associated with a100%bootstrap value. TetM was shown to be identical to previously described TetM with a97%bootstrap value. Lb. crispatus K243and K313were used as the donor strains, and Enterococcus faecalis K9as the receptor strain. All the resistence genes could not transfer into Enterococcus faecalis K9, indicating that the resistence genes were stable in Lb. crispatus K243and K313.5. Construction of expression vectors for LactobacillusIn vitro evaluations of the probiotic role of Lb. crispatus K243and K313were performed in previous research. In order to study their colonizations and dynamic changes of growth in vivo, fluorescently labeled plasmid vector carrying the fluorescent protein gene was proposed to transform into Lactobacillus. The molecular tools for the genetic manipulation of lactobacilli are still not consummate. Based on this, a cryptic plasmid pD403was isolated from Lactobacillus plantarum D403derived from fermented dairy products in this work. It was2,791bp in size with a G+C content of37%. Nucleotide sequence analysis revealed two open reading frames, orfl and orf2. ORF1(318amino acids) was identified as a replication protein (RepA). ORF2(137amino acids) shared31%identity with the transcriptional regulator of Ralstonia pickettii12D. Functional investigation indicated that ORF2(Tra) is unessential for replication in different hosts, but it had the ability of improving the transformation efficiency. The origin of replication was predicted, suggesting that pD403was a rolling-circle-replication (RCR) plasmid, and The RepA had the high identity with the replication initiation protein belonged to group III family. To develop molecular tools on the basis of pD403, the replicon of pUC19, chloramphenicol resistant gene and the repA from pD403were ligated subsequently, yielding an E. coli/Lactobacillus shuttle vector pCD4032. The host range of the vector pCD4032was determined, and Lb. casei BL23, Lb. plantarum LPA, Lb. casei401, Lb. fermentum YB5and Lb. brevis CGMCC1.208could be transformed successfully. The transformation efficiencies were ranged from1.3×102to7×104transformants per microgram DNA. According to the genome sequence of the Lb. delbrueckii subsp. bulgaricus ATCC11842, the promoter and terminator sequences from lactate dehydrogenase were amplified and cloned into pCD4032, generating the expression vector pCD4033. Gfp gene was inserted into the downstream of the promoter pldh to generate the recombinant plasmid pCD4033-gfp. Then the plasmid pCD4033-gfp was electroporated into various lactobacilli cells. The green fluorescence in most of recombinant cells was observed. however, no fluorescence was observed in control strains. After optimizing the expression conditions, the highest amount of fluorescence was obtained when the transformants were incubated with aeration at30℃. These results proved that GFP was expressed successfully, and the vector pCD4033was feasible for heterologous proteins expression in Lactobacillus strains. Unfortunately, The vector pCD4033can not transform into Lb. crispatus K243and K313.更多还原