卷曲乳杆菌益生特性及黏附机理研究
|
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。
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 strains
Currently, 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-29cells
Probiotics 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 K313
It 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. crispatus
In 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 Lactobacillus
In 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.
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。
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 strains
Currently, 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-29cells
Probiotics 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 K313
It 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. crispatus
In 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 Lactobacillus
In 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.
引文
Alander, M., Satokari, R., Korpela, R., Saxelin, M., Vilpponen-Salmela, T. Mattila-Sandholm, T., von Wright, A.,1999, Persistence of colonization of human colonic mucosa by a probiotic strain, Lactobacillus rhamnosus GG, after oral consumption. Appl Environ Microbiol 65,351-354.
Ames, B.N., Dubin, D.T.,1960, The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem 235,769-775.
An, H.Y., Miyamoto, T.,2006, Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei:construction of an Escherichia coli-Lactobacillus shuttle vector. Plasmid 55,128-134.
Antikainen, J., Anton, L., Sillanpaa, J., Korhonen, T.K.,2002, Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly. Mol Microbiol 46, 381-394.
Avall-Jaaskelainen, S., Hynonen, U., Ilk, N., Pum, D., Sleytr, U.B., Palva, A.,2008, Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287. BMC Microbiol 8,165.
Avall-Jaaskelainen, S., Kyla-Nikkila, K., Kahala, M., Miikkulainen-Lahti, T., Palva, A.,2002, Surface display of foreign epitopes on the Lactobacillus brevis S-layer. Appl Environ Microbiol 68,5943-5951.
Avall-Jaaskelainen, S., Lindholm, A., Palva, A.,2003, Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells. Appl Environ Microbiol 69,2230-2236.
Avall-Jaaskelainen, S., Palva, A.,2005, Lactobacillus surface layers and their applications. FEMS Microbiol Rev 29,511-529.
Bai, A.P., Ouyang, Q., Xiao, X.R., Li, S.F.,2006, Probiotics modulate inflammatory cytokine secretion from inflamed mucosa in active ulcerative colitis. Int J Clin Pract 60,284-288.
Bernardeau, M., Vernoux, J.P., Henri-Dubernet, S., Gueguen, M.,2008, Safety assessment of dairy microorganisms:the Lactobacillus genus. Int J Food Microbiol 126,278-285.
Bernet, M.F., Brassart, D., Neeser, J.R., Servin, A.L.,1994, Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 35,483-489.
Bezkorovainy, A.,2001, Probiotics:determinants of survival and growth in the gut. Am J Clin Nutr 73,399S-405S.
Boot, H.J., Kolen, C.P., Andreadaki, F.J., Leer, R.J., Pouwels, P.H.,1996a, The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J Bacteriol 178,5388-5394.
Boot, H.J., Kolen, C.P., Pot, B., Kersters, K., Pouwels, P.H.,1996b, The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142 (Pt 9),2375-2384.
Boot, H.J., Kolen, C.P., van Noort, J.M., Pouwels, P.H.,1993, S-layer protein of Lactobacillus acidophilus ATCC 4356:purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J Bacteriol 175, 6089-6096.
Bouia, A., Bringel, F., Frey, L., Kammerer, B., Belarbi, A., Guyonvarch, A., Hubert, J.C.,1989, Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904. Plasmid 22,185-192.
Buck, B.L., Altermann, E., Svingerud, T., Klaenhammer, T.R.,2005, Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71,8344-8351.
Callegari, M.L., Riboli, B., Sanders, J.W., Cocconcelli, P.S., Kok, J., Venema, G., Morelli, L.,1998, The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression. Microbiology 144 (Pt 3), 719-726.
Cataloluk, O., Gogebakan, B.,2004, Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey. FEMS Microbiol Lett 236, 7-12.
Chagnaud, P., Chan Kwo Chion, C.K., Duran, R., Naouri, P., Arnaud, A., Galzy, P., 1992, Construction of a new shuttle vector for Lactobacillus. Can J Microbiol 38,69-74.
Chen, X., Chen, Y., Li, X., Chen, N., Fang, W.,2009, Characterization of surface layer proteins in Lactobacillus crispatus isolate ZJ001. J Microbiol Biotechnol 19,1176-1183.
Chen, X., Kokkotou, E.G., Mustafa, N., Bhaskar, K.R., Sougioultzis, S., O'Brien, M., Pothoulakis, C., Kelly, C.P.,2006, Saccharomyces boulardii inhibits ERK1/2 mitogen-activated protein kinase activation both in vitro and in vivo and protects against Clostridium difficile toxin A-induced enteritis. J Biol Chem 281,24449-24454.
Chen, X., Xu, J., Shuai, J., Chen, J., Zhang, Z., Fang, W.,2007, The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium. Int J Food Microbiol 115,307-312.
Chiu, C.H., Lu, T.Y., Tseng, Y.Y., Pan, T.M.,2006, The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet. Appl Microbiol Biotechnol 71,238-245.
Claesson, M.J., Li, Y., Leahy, S., Canchaya, C., van Pijkeren, J.P., Cerdeno-Tarraga, A.M., Parkhill, J., Flynn, S., O'Sullivan, G.C., Collins, J.K., Higgins, D., Shanahan, F., Fitzgerald, G.F., van Sinderen, D., O'Toole, P.W.,2006, Multireplicon genome architecture of Lactobacillus salivarius. Proc Natl Acad Sci U S A 103,6718-6723.
Courvalin, P.,1994, Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Antimicrob Agents Chemother 38,1447-1451.
Del Re, B., Sgorbati, B., Miglioli, M., Palenzona, D.,2000, Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31,438-442.
Dubuquoy, L., Jansson, E.A., Deeb, S., Rakotobe, S., Karoui, M., Colombel, J.F., Auwerx, J., Pettersson, S., Desreumaux, P.,2003, Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis. Gastroenterology 124,1265-1276.
Felis, G.E., Dellaglio, F.,2007, Taxonomy of Lactobacilli and Bifidobacteria. Curr Issues Intest Microbiol 8,44-61.
Fons, M., Hege, T., Ladire, M., Raibaud, P., Ducluzeau, R., Maguin, E.,1997, Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance. Plasmid 37,199-203.
Frece, J., Kos, B., Svetec, I.K., Zgaga, Z., Mrsa, V., Suskovic, J.,2005, Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92. J Appl Microbiol 98,285-292.
Frick, J.S., Schenk, K., Quitadamo, M., Kahl, F., Koberle, M., Bohn, E., Aepfelbacher, M., Autenrieth, I.B.,2007, Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells. Inflamm Bowel Dis 13,83-90.
Fuller, R.,1989, Probiotics in man and animals. J Appl Bacteriol 66,365-378.
Gilliland, S.E.,1989, Acidophilus milk products:a review of potential benefits to consumers. J Dairy Sci 72,2483-2494.
Goh, Y.J., Azcarate-Peril, M.A., O'Flaherty, S., Durmaz, E., Valence, F., Jardin, J., Lortal, S., Klaenhammer, T.R.,2009, Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 75,3093-3105.
Golowczyc, M.A., Mobili, P., Garrote, G.L., Abraham, A.G., De Antoni, G.L.,2007, Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis. Int J Food Microbiol 118,264-273.
Graves, M.C., Rabinowitz, J.C.,1986, In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem 261,11409-11415.
Guimaraes, V.D., Innocentin, S., Lefevre, F., Azevedo, V., Wal, J.M., Langella, P., Chatel, J.M.,2006, Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells. Appl Environ Microbiol 72,7091-7097.
Gusils, C., Cuozzo, S., Sesma, F., Gonzalez, S.,2002, Examination of adhesive determinants in three species of Lactobacillus isolated from chicken. Can J Microbiol 48,34-42.
Hagen, K.E., Guan, L.L., Tannock, G.W., Korver, D.R., Allison, G.E.,2005, Detection, characterization, and in vitro and in vivo expression of genes encoding S-proteins in Lactobacillus gallinarum strains isolated from chicken crops. Appl Environ Microbiol 71,6633-6643.
Haller, D., Russo, M.P., Sartor, R.B., Jobin, C.,2002, IKK beta and phosphatidylinositol 3-kinase/Akt participate in non-pathogenic Gram-negative enteric bacteria-induced RelA phosphorylation and NF-kappa B activation in both primary and intestinal epithelial cell lines. J Biol Chem 277,38168-38178.
Hazebrouck, S., Pothelune, L., Azevedo, V., Corthier, G., Wal, J.M., Langella, P., 2007, Efficient production and secretion of bovine beta-lactoglobulin by Lactobacillus casei. Microb Cell Fact 6,12.
He, F., Ouwehan, A.C., Hashimoto, H., Isolauri, E., Benno, Y., Salminen, S.,2001a, Adhesion of Bifidobacterium spp. to human intestinal mucus. Microbiol Immunol 45,259-262.
He, F., Ouwehand, A.C., Isolauri, E., Hashimoto, H., Benno, Y., Salminen, S.,2001b, Comparison of mucosal adhesion and species identification of bifidobacteria isolated from healthy and allergic infants. FEMS Immunol Med Microbiol 30, 43-47.
Heng, N.C., Bateup, J.M., Loach, D.M., Wu, X., Jenkinson, H.F., Morrison, M., Tannock, G.W.,1999, Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo. Appl Environ Microbiol 65,5378-5385.
Horie, M., Kajikawa, H.S., Toba, T.,2002, Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene. Lett Appl Microbiol 35,57-61.
Huber, C., Ilk, N., Runzler, D., Egelseer, E.M., Weigert, S., Sleytr, U.B., Sara, M., 2005, The three S-layer-like homology motifs of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer. Mol Microbiol 55,197-205.
Hudault, S., Lievin, V., Bernet-Camard, M.F., Servin, A.L.,1997, Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection. Appl Environ Microbiol 63,513-518.
Hynonen, U., Avall-Jaaskelainen, S., Palva, A.,2010, Characterization and separate activities of the two promoters of the Lactobacillus brevis S-layer protein gene. Appl Microbiol Biotechnol 87,657-668.
Ishibashi, N., Yamazaki, S.,2001, Probiotics and safety. Am J Clin Nutr 73, 465S-470S.
Jakava-Viljanen, M., Avall-Jaaskelainen, S., Messner, P., Sleytr, U.B., Palva, A., 2002, Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions. J Bacteriol 184, 6786-6795.
Jakava-Viljanen, M., Palva, A.,2007, Isolation of surface (S) layer protein carrying Lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues. Vet Microbiol 124,264-273.
Johnson-Henry, K.C., Hagen, K.E., Gordonpour, M., Tompkins, T.A., Sherman, P.M., 2007, Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells. Cell Microbiol 9,356-367.
Kahala, M., Palva, A.,1999, The expression signals of the Lactobacillus brevis slpA gene direct efficient heterologous protein production in lactic acid bacteria. Appl Microbiol Biotechnol 51,71-78.
Kahala, M., Savijoki, K., Palva, A.,1997, In vivo expression of the Lactobacillus brevis S-layer gene. J Bacteriol 179,284-286.
Kaneko, Y., Kobayashi, H., Kiatpapan, P., Nishimoto, T., Napitupulu, R., Ono, H., Murooka, Y.,2000, Development of a host-vector system for Lactobacillus plantarum L137 isolated from a traditional fermented food produced in the Philippines. J Biosci Bioeng 89,62-67.
Khan, S.A.,1997, Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev 61,442-455.
Konstantinov, S.R., Smidt, H., de Vos, W.M., Bruijns, S.C., Singh, S.K., Valence, F., Molle, D., Lortal, S., Altermann, E., Klaenhammer, T.R., van Kooyk, Y.,2008, S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions. Proc Natl Acad Sci U S A 105, 19474-19479.
Kos, B., Suskovic, J., Vukovic, S., Simpraga, M., Frece, J., Matosic, S.,2003, Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94,981-987.
Kumar, A., Wu, H., Collier-Hyams, L.S., Hansen, J.M., Li, T., Yamoah, K., Pan, Z.Q., Jones, D.P., Neish, A.S.,2007, Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species. EMBO J 26,4457-4466.
Lee, J.H., Halgerson, J.S., Kim, J.H., O'Sullivan, D.J.,2007, Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector. Appl Environ Microbiol 73,4417-4424.
Leer, R.J., van Luijk, N., Posno, M., Pouwels, P.H.,1992, Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol Gen Genet 234,265-274.
Lilly, D.M., Stillwell, R.H.,1965, Probiotics:Growth-Promoting Factors Produced by Microorganisms. Science 147,747-748.
Lin, P.W., Myers, L.E., Ray, L., Song, S.C., Nasr, T.R., Berardinelli, A.J., Kundu, K., Murthy, N., Hansen, J.M., Neish, A.S.,2009, Lactobacillus rhamnosus blocks inflammatory signaling in vivo via reactive oxygen species generation. Free Radic Biol Med 47,1205-1211.
Liu, Y.G., Whittier, R.F.,1995, Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25,674-681.
Llopis, M., Antolin, M., Carol, M., Borruel, N., Casellas, F., Martinez, C., Espin-Basany, E., Guarner, F., Malagelada, J.R.,2009, Lactobacillus casei downregulates commensals'inflammatory signals in Crohn's disease mucosa. Inflamm Bowel Dis 15,275-283.
Lokman, B.C., van Santen, P., Verdoes, J.C., Kruse, J., Leer, R.J., Posno, M., Pouwels, P.H.,1991, Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus. Mol Gen Genet 230,161-169.
Ma, D., Forsythe, P., Bienenstock, J.,2004, Live Lactobacillus rhamnosus is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression. Infect Immun 72,5308-5314.
Mack, D.R., Ahrne, S., Hyde, L., Wei, S., Hollingsworth, M.A.,2003, Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut 52,827-833.
Marraffini, L.A., Dedent, A.C., Schneewind, O.,2006, Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70,192-221.
Mierau, I., Kleerebezem, M.,2005,10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68,705-717.
Narita, J., Ishida, S., Okano, K., Kimura, S., Fukuda, H., Kondo, A.,2006, Improvement of protein production in lactic acid bacteria using 5'-untranslated leader sequence of slpA from Lactobacillus acidophilus. Appl Microbiol Biotechnol 73,366-373.
Neish, A.S., Gewirtz, A.T., Zeng, H., Young, A.N., Hobert, M.E., Karmali, V., Rao, A.S., Madara, J.L.,2000, Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science 289,1560-1563.
O'Hara, A.M., O'Regan, P., Fanning, A., O'Mahony, C., Macsharry, J., Lyons, A., Bienenstock, J., O'Mahony, L., Shanahan, F.,2006, Functional modulation of human intestinal epithelial cell responses by Bifidobacterium infantis and Lactobacillus salivarius. Immunology 118,202-215.
Oozeer, R., Furet, J.P., Goupil-Feuillerat, N., Anba, J., Mengaud, J., Corthier, G., 2005, Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice. Appl Environ Microbiol 71,1356-1363.
Oozeer, R., Goupil-Feuillerat, N., Alpert, C.A., van de Guchte, M., Anba, J., Mengaud, J., Corthier, G.,2002, Lactobacillus casei is able to survive and initiate protein synthesis during its transit in the digestive tract of human flora-associated mice. Appl Environ Microbiol 68,3570-3574.
Ouwehand, A.C., Tuomola, E.M., Tolkko, S., Salminen, S.,2001, Assessment of adhesion properties of novel probiotic strains to human intestinal mucus. Int J Food Microbiol 64,119-126.
Pelletier, C., Bouley, C., Cayuela, C., Bouttier, S., Bourlioux, P., Bellon-Fontaine, M.N.,1997, Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains. Appl Environ Microbiol 63,1725-1731.
Perez-Arellano, I., Perez-Martinez, G.,2003, Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei. FEMS Microbiol Lett 222,123-127.
Petrof, E.O., Claud, E.C., Sun, J., Abramova, T., Guo, Y., Waypa, T.S., He, S.M., Nakagawa, Y., Chang, E.B.,2009, Bacteria-free solution derived from Lactobacillus plantarum inhibits multiple NF-kappaB pathways and inhibits proteasome function. Inflamm Bowel Dis 15,1537-1547.
Pouwels, P.H., Leer, R.J.,1993, Genetics of lactobacilli:plasmids and gene expression. Antonie Van Leeuwenhoek 64,85-107.
Pouwels, P.H., van Luijk, N., Leer, R.J., Posno, M.,1994, Control of replication of the Lactobacillus pentosus plasmid p353-2:evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein. Mol Gen Genet 242,614-622.
Pretzer, G., Snel, J., Molenaar, D., Wiersma, A., Bron, P.A., Lambert, J., de Vos, W.M., van der Meer, R., Smits, M.A., Kleerebezem, M.,2005, Biodiversity-based identification and functional characterization of the mannose-specific adhesin of Lactobacillus plantarum. J Bacteriol 187, 6128-6136.
Reid, G., Burton, J.,2002, Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4,319-324.
Resta-Lenert, S., Barrett, K.E.,2003, Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC). Gut 52,988-997.
Resta-Lenert, S., Barrett, K.E.,2006, Probiotics and commensals reverse TNF-alpha-and IFN-gamma-induced dysfunction in human intestinal epithelial cells. Gastroenterology 130,731-746.
Rinkinen, M., Westermarck, E., Salminen, S., Ouwehand, A.C.,2003, Absence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus. Vet Microbiol 97,55-61.
Rojas, M., Ascencio, F., Conway, P.L.,2002, Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin. Appl Environ Microbiol 68, 2330-2336.
Ruiz, P.A., Hoffmann, M., Szcesny, S., Blaut, M., Haller, D.,2005, Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats. Immunology 115,441-450.
Saarela, M., Mogensen, G., Fonden, R., Matto, J., Mattila-Sandholm, T.,2000, Probiotic bacteria:safety, functional and technological properties. J Biotechnol 84,197-215.
Schlee, M., Harder, J., Koten, B., Stange, E.F., Wehkamp, J., Fellermann, K.,2008, Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp Immunol 151,528-535.
Schmieger, H., Schicklmaier, P.,1999, Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104. FEMS Microbiol Lett 170, 251-256.
Seth, A., Yan, F., Polk, D.B., Rao, R.K.,2008, Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC-and MAP kinase-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 294, G1060-1069.
Sillanpaa, J., Martinez, B., Antikainen, J., Toba, T., Kalkkinen, N., Tankka, S., Lounatmaa, K., Keranen, J., Hook, M., Westerlund-Wikstrom, B., Pouwels, P.H., Korhonen, T.K.,2000, Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus. J Bacteriol 182,6440-6450.
Smit, E., Jager, D., Martinez, B., Tielen, F.J., Pouwels, P.H.,2002, Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356:evidence for protein-protein interaction of two subdomains. J Mol Biol 324,953-964.
Smit, E., Oling, F., Demel, R., Martinez, B., Pouwels, P.H.,2001, The S-layer protein of Lactobacillus acidophilus ATCC 4356:identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol 305,245-257.
Smit, E., Pouwels, P.H.,2002, One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein. J Bacteriol 184,4617-4619.
Sorvig, E., Skaugen, M., Naterstad, K., Eijsink, V.G., Axelsson, L.,2005b, Plasmid p256 from Lactobacillus plantarum represents a new type of replicon in lactic acid bacteria, and contains a toxin-antitoxin-like plasmid maintenance system. Microbiology 151,421-431.
Tao, Y., Drabik, K.A., Waypa, T.S., Musch, M.W., Alverdy, J.C., Schneewind, O., Chang, E.B., Petrof, E.O.,2006, Soluble factors from Lactobacillus GG activate MAPKs and induce cytoprotective heat shock proteins in intestinal epithelial cells. Am J Physiol Cell Physiol 290, C1018-1030.
Tassell, M.L., Miller, M.J.,2011, Lactobacillus adhesion to mucus. Nutrients 3, 613-636.
Tien, M.T., Girardin, S.E., Regnault, B., Le Bourhis, L., Dillies, M.A., Coppee, J.Y., Bourdet-Sicard, R., Sansonetti, P.J., Pedron, T.,2006, Anti-inflammatory effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells. J Immunol 176,1228-1237.
Tiurin, M.V.,1992, [Electrotransformation of bacteria]. Antibiot Khimioter 37,51-55.
Tuomola, E., Crittenden, R., Playne, M., Isolauri, E., Salminen, S.,2001, Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 73,393S-398S.
Vadillo-Rodriguez, V., Busscher, H.J., Norde, W., de Vries, J., van der Mei, H.C., 2004, Dynamic cell surface hydrophobicity of Lactobacillus strains with and without surface layer proteins. J Bacteriol 186,6647-6650.
van de Guchte, M., Penaud, S., Grimaldi, C., Barbe, V., Bryson, K., Nicolas, P., Robert, C., Oztas, S., Mangenot, S., Couloux, A., Loux, V., Dervyn, R., Bossy, R., Bolotin, A., Batto, J.M., Walunas, T., Gibrat, J.F., Bessieres, P., Weissenbach, J., Ehrlich, S.D., Maguin, E.,2006, The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A 103,9274-9279.
van Pijkeren, J.P., Canchaya, C., Ryan, K.A., Li, Y., Claesson, M.J., Sheil, B., Steidler, L., O'Mahony, L., Fitzgerald, G.F., van Sinderen, D., O'Toole, P.W., 2006, Comparative and functional analysis of sortase-dependent proteins in the predicted secretome of Lactobacillus salivarius UCC118. Appl Environ Microbiol 72,4143-4153.
Velez, M.P., De Keersmaecker, S.C., Vanderleyden, J.,2007, Adherence factors of Lactobacillus in the human gastrointestinal tract. FEMS Microbiol Lett 276, 140-148.
Ventura, M., Jankovic, I., Walker, D.C., Pridmore, R.D., Zink, R.,2002, Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri. Appl Environ Microbiol 68,6172-6181.
Vidgren, G., Palva, I., Pakkanen, R., Lounatmaa, K., Palva, A.,1992, S-layer protein gene of Lactobacillus brevis:cloning by polymerase chain reaction and determination of the nucleotide sequence. J Bacteriol 174,7419-7427.
Voltan, S., Martines, D., Elli, M., Brun, P., Longo, S., Porzionato, A., Macchi, V., D'Inca, R., Scarpa, M., Palu, G., Sturniolo, G.C., Morelli, L., Castagliuolo, I., 2008, Lactobacillus crispatus M247-derived H2O2 acts as a signal transducing molecule activating peroxisome proliferator activated receptor-gamma in the intestinal mucosa. Gastroenterology 135,1216-1227.
Vujcic, M., Topisirovic, L.,1993, Molecular analysis of the rolling-circle replicating plasmid pAl of Lactobacillus plantarum A112. Appl Environ Microbiol 59, 274-280.
Wu, G.D., Huang, N., Wen, X., Keilbaugh, S.A., Yang, H.,1999, High-level expression of I kappa B-beta in the surface epithelium of the colon:in vitro evidence for an immunomodulatory role. J Leukoc Biol 66,1049-1056.
Zhang, L., Li, N., Caicedo, R., Neu, J.,2005, Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factor-alpha-induced interleukin-8 production in Caco-2 cells. J Nutr 135,1752-1756.
Zhang, L., Xu, Y.Q., Liu, H.Y., Lai, T., Ma, J.L., Wang, J.F., Zhu, Y.H.,2010, Evaluation of Lactobacillus rhamnosus GG using an Escherichia coli K88 model of piglet diarrhoea:Effects on diarrhoea incidence, faecal microflora and immune responses. Vet Microbiol 141,142-148.
蔡凯凯,黄占旺,叶德军,孙长涛.益生菌调节肠道菌群及免疫调节作用机理[J].中国饲料,2011,18：34-37.
刘延国,张琳,冯香安,李杰.益生菌在消化道中的抑菌作用[J].中国饲料,2011,16：9-12.
罗丽娟,施季森.一种DNA侧翼序列分离技术——TAIL-PCR[J].南京林业大学学报,2003,27：87-90.
Ames, B.N., Dubin, D.T.,1960, The role of polyamines in the neutralization of bacteriophage deoxyribonucleic acid. J Biol Chem 235,769-775.
An, H.Y., Miyamoto, T.,2006, Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei:construction of an Escherichia coli-Lactobacillus shuttle vector. Plasmid 55,128-134.
Antikainen, J., Anton, L., Sillanpaa, J., Korhonen, T.K.,2002, Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly. Mol Microbiol 46, 381-394.
Avall-Jaaskelainen, S., Hynonen, U., Ilk, N., Pum, D., Sleytr, U.B., Palva, A.,2008, Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287. BMC Microbiol 8,165.
Avall-Jaaskelainen, S., Kyla-Nikkila, K., Kahala, M., Miikkulainen-Lahti, T., Palva, A.,2002, Surface display of foreign epitopes on the Lactobacillus brevis S-layer. Appl Environ Microbiol 68,5943-5951.
Avall-Jaaskelainen, S., Lindholm, A., Palva, A.,2003, Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells. Appl Environ Microbiol 69,2230-2236.
Avall-Jaaskelainen, S., Palva, A.,2005, Lactobacillus surface layers and their applications. FEMS Microbiol Rev 29,511-529.
Bai, A.P., Ouyang, Q., Xiao, X.R., Li, S.F.,2006, Probiotics modulate inflammatory cytokine secretion from inflamed mucosa in active ulcerative colitis. Int J Clin Pract 60,284-288.
Bernardeau, M., Vernoux, J.P., Henri-Dubernet, S., Gueguen, M.,2008, Safety assessment of dairy microorganisms:the Lactobacillus genus. Int J Food Microbiol 126,278-285.
Bernet, M.F., Brassart, D., Neeser, J.R., Servin, A.L.,1994, Lactobacillus acidophilus LA 1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria. Gut 35,483-489.
Bezkorovainy, A.,2001, Probiotics:determinants of survival and growth in the gut. Am J Clin Nutr 73,399S-405S.
Boot, H.J., Kolen, C.P., Andreadaki, F.J., Leer, R.J., Pouwels, P.H.,1996a, The Lactobacillus acidophilus S-layer protein gene expression site comprises two consensus promoter sequences, one of which directs transcription of stable mRNA. J Bacteriol 178,5388-5394.
Boot, H.J., Kolen, C.P., Pot, B., Kersters, K., Pouwels, P.H.,1996b, The presence of two S-layer-protein-encoding genes is conserved among species related to Lactobacillus acidophilus. Microbiology 142 (Pt 9),2375-2384.
Boot, H.J., Kolen, C.P., van Noort, J.M., Pouwels, P.H.,1993, S-layer protein of Lactobacillus acidophilus ATCC 4356:purification, expression in Escherichia coli, and nucleotide sequence of the corresponding gene. J Bacteriol 175, 6089-6096.
Bouia, A., Bringel, F., Frey, L., Kammerer, B., Belarbi, A., Guyonvarch, A., Hubert, J.C.,1989, Structural organization of pLP1, a cryptic plasmid from Lactobacillus plantarum CCM 1904. Plasmid 22,185-192.
Buck, B.L., Altermann, E., Svingerud, T., Klaenhammer, T.R.,2005, Functional analysis of putative adhesion factors in Lactobacillus acidophilus NCFM. Appl Environ Microbiol 71,8344-8351.
Callegari, M.L., Riboli, B., Sanders, J.W., Cocconcelli, P.S., Kok, J., Venema, G., Morelli, L.,1998, The S-layer gene of Lactobacillus helveticus CNRZ 892: cloning, sequence and heterologous expression. Microbiology 144 (Pt 3), 719-726.
Cataloluk, O., Gogebakan, B.,2004, Presence of drug resistance in intestinal lactobacilli of dairy and human origin in Turkey. FEMS Microbiol Lett 236, 7-12.
Chagnaud, P., Chan Kwo Chion, C.K., Duran, R., Naouri, P., Arnaud, A., Galzy, P., 1992, Construction of a new shuttle vector for Lactobacillus. Can J Microbiol 38,69-74.
Chen, X., Chen, Y., Li, X., Chen, N., Fang, W.,2009, Characterization of surface layer proteins in Lactobacillus crispatus isolate ZJ001. J Microbiol Biotechnol 19,1176-1183.
Chen, X., Kokkotou, E.G., Mustafa, N., Bhaskar, K.R., Sougioultzis, S., O'Brien, M., Pothoulakis, C., Kelly, C.P.,2006, Saccharomyces boulardii inhibits ERK1/2 mitogen-activated protein kinase activation both in vitro and in vivo and protects against Clostridium difficile toxin A-induced enteritis. J Biol Chem 281,24449-24454.
Chen, X., Xu, J., Shuai, J., Chen, J., Zhang, Z., Fang, W.,2007, The S-layer proteins of Lactobacillus crispatus strain ZJ001 is responsible for competitive exclusion against Escherichia coli O157:H7 and Salmonella typhimurium. Int J Food Microbiol 115,307-312.
Chiu, C.H., Lu, T.Y., Tseng, Y.Y., Pan, T.M.,2006, The effects of Lactobacillus-fermented milk on lipid metabolism in hamsters fed on high-cholesterol diet. Appl Microbiol Biotechnol 71,238-245.
Claesson, M.J., Li, Y., Leahy, S., Canchaya, C., van Pijkeren, J.P., Cerdeno-Tarraga, A.M., Parkhill, J., Flynn, S., O'Sullivan, G.C., Collins, J.K., Higgins, D., Shanahan, F., Fitzgerald, G.F., van Sinderen, D., O'Toole, P.W.,2006, Multireplicon genome architecture of Lactobacillus salivarius. Proc Natl Acad Sci U S A 103,6718-6723.
Courvalin, P.,1994, Transfer of antibiotic resistance genes between gram-positive and gram-negative bacteria. Antimicrob Agents Chemother 38,1447-1451.
Del Re, B., Sgorbati, B., Miglioli, M., Palenzona, D.,2000, Adhesion, autoaggregation and hydrophobicity of 13 strains of Bifidobacterium longum. Lett Appl Microbiol 31,438-442.
Dubuquoy, L., Jansson, E.A., Deeb, S., Rakotobe, S., Karoui, M., Colombel, J.F., Auwerx, J., Pettersson, S., Desreumaux, P.,2003, Impaired expression of peroxisome proliferator-activated receptor gamma in ulcerative colitis. Gastroenterology 124,1265-1276.
Felis, G.E., Dellaglio, F.,2007, Taxonomy of Lactobacilli and Bifidobacteria. Curr Issues Intest Microbiol 8,44-61.
Fons, M., Hege, T., Ladire, M., Raibaud, P., Ducluzeau, R., Maguin, E.,1997, Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance. Plasmid 37,199-203.
Frece, J., Kos, B., Svetec, I.K., Zgaga, Z., Mrsa, V., Suskovic, J.,2005, Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92. J Appl Microbiol 98,285-292.
Frick, J.S., Schenk, K., Quitadamo, M., Kahl, F., Koberle, M., Bohn, E., Aepfelbacher, M., Autenrieth, I.B.,2007, Lactobacillus fermentum attenuates the proinflammatory effect of Yersinia enterocolitica on human epithelial cells. Inflamm Bowel Dis 13,83-90.
Fuller, R.,1989, Probiotics in man and animals. J Appl Bacteriol 66,365-378.
Gilliland, S.E.,1989, Acidophilus milk products:a review of potential benefits to consumers. J Dairy Sci 72,2483-2494.
Goh, Y.J., Azcarate-Peril, M.A., O'Flaherty, S., Durmaz, E., Valence, F., Jardin, J., Lortal, S., Klaenhammer, T.R.,2009, Development and application of a upp-based counterselective gene replacement system for the study of the S-layer protein SlpX of Lactobacillus acidophilus NCFM. Appl Environ Microbiol 75,3093-3105.
Golowczyc, M.A., Mobili, P., Garrote, G.L., Abraham, A.G., De Antoni, G.L.,2007, Protective action of Lactobacillus kefir carrying S-layer protein against Salmonella enterica serovar Enteritidis. Int J Food Microbiol 118,264-273.
Graves, M.C., Rabinowitz, J.C.,1986, In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene. Evidence for "extended" promoter elements in gram-positive organisms. J Biol Chem 261,11409-11415.
Guimaraes, V.D., Innocentin, S., Lefevre, F., Azevedo, V., Wal, J.M., Langella, P., Chatel, J.M.,2006, Use of native lactococci as vehicles for delivery of DNA into mammalian epithelial cells. Appl Environ Microbiol 72,7091-7097.
Gusils, C., Cuozzo, S., Sesma, F., Gonzalez, S.,2002, Examination of adhesive determinants in three species of Lactobacillus isolated from chicken. Can J Microbiol 48,34-42.
Hagen, K.E., Guan, L.L., Tannock, G.W., Korver, D.R., Allison, G.E.,2005, Detection, characterization, and in vitro and in vivo expression of genes encoding S-proteins in Lactobacillus gallinarum strains isolated from chicken crops. Appl Environ Microbiol 71,6633-6643.
Haller, D., Russo, M.P., Sartor, R.B., Jobin, C.,2002, IKK beta and phosphatidylinositol 3-kinase/Akt participate in non-pathogenic Gram-negative enteric bacteria-induced RelA phosphorylation and NF-kappa B activation in both primary and intestinal epithelial cell lines. J Biol Chem 277,38168-38178.
Hazebrouck, S., Pothelune, L., Azevedo, V., Corthier, G., Wal, J.M., Langella, P., 2007, Efficient production and secretion of bovine beta-lactoglobulin by Lactobacillus casei. Microb Cell Fact 6,12.
He, F., Ouwehan, A.C., Hashimoto, H., Isolauri, E., Benno, Y., Salminen, S.,2001a, Adhesion of Bifidobacterium spp. to human intestinal mucus. Microbiol Immunol 45,259-262.
He, F., Ouwehand, A.C., Isolauri, E., Hashimoto, H., Benno, Y., Salminen, S.,2001b, Comparison of mucosal adhesion and species identification of bifidobacteria isolated from healthy and allergic infants. FEMS Immunol Med Microbiol 30, 43-47.
Heng, N.C., Bateup, J.M., Loach, D.M., Wu, X., Jenkinson, H.F., Morrison, M., Tannock, G.W.,1999, Influence of different functional elements of plasmid pGT232 on maintenance of recombinant plasmids in Lactobacillus reuteri populations in vitro and in vivo. Appl Environ Microbiol 65,5378-5385.
Horie, M., Kajikawa, H.S., Toba, T.,2002, Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene. Lett Appl Microbiol 35,57-61.
Huber, C., Ilk, N., Runzler, D., Egelseer, E.M., Weigert, S., Sleytr, U.B., Sara, M., 2005, The three S-layer-like homology motifs of the S-layer protein SbpA of Bacillus sphaericus CCM 2177 are not sufficient for binding to the pyruvylated secondary cell wall polymer. Mol Microbiol 55,197-205.
Hudault, S., Lievin, V., Bernet-Camard, M.F., Servin, A.L.,1997, Antagonistic activity exerted in vitro and in vivo by Lactobacillus casei (strain GG) against Salmonella typhimurium C5 infection. Appl Environ Microbiol 63,513-518.
Hynonen, U., Avall-Jaaskelainen, S., Palva, A.,2010, Characterization and separate activities of the two promoters of the Lactobacillus brevis S-layer protein gene. Appl Microbiol Biotechnol 87,657-668.
Ishibashi, N., Yamazaki, S.,2001, Probiotics and safety. Am J Clin Nutr 73, 465S-470S.
Jakava-Viljanen, M., Avall-Jaaskelainen, S., Messner, P., Sleytr, U.B., Palva, A., 2002, Isolation of three new surface layer protein genes (slp) from Lactobacillus brevis ATCC 14869 and characterization of the change in their expression under aerated and anaerobic conditions. J Bacteriol 184, 6786-6795.
Jakava-Viljanen, M., Palva, A.,2007, Isolation of surface (S) layer protein carrying Lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues. Vet Microbiol 124,264-273.
Johnson-Henry, K.C., Hagen, K.E., Gordonpour, M., Tompkins, T.A., Sherman, P.M., 2007, Surface-layer protein extracts from Lactobacillus helveticus inhibit enterohaemorrhagic Escherichia coli O157:H7 adhesion to epithelial cells. Cell Microbiol 9,356-367.
Kahala, M., Palva, A.,1999, The expression signals of the Lactobacillus brevis slpA gene direct efficient heterologous protein production in lactic acid bacteria. Appl Microbiol Biotechnol 51,71-78.
Kahala, M., Savijoki, K., Palva, A.,1997, In vivo expression of the Lactobacillus brevis S-layer gene. J Bacteriol 179,284-286.
Kaneko, Y., Kobayashi, H., Kiatpapan, P., Nishimoto, T., Napitupulu, R., Ono, H., Murooka, Y.,2000, Development of a host-vector system for Lactobacillus plantarum L137 isolated from a traditional fermented food produced in the Philippines. J Biosci Bioeng 89,62-67.
Khan, S.A.,1997, Rolling-circle replication of bacterial plasmids. Microbiol Mol Biol Rev 61,442-455.
Konstantinov, S.R., Smidt, H., de Vos, W.M., Bruijns, S.C., Singh, S.K., Valence, F., Molle, D., Lortal, S., Altermann, E., Klaenhammer, T.R., van Kooyk, Y.,2008, S layer protein A of Lactobacillus acidophilus NCFM regulates immature dendritic cell and T cell functions. Proc Natl Acad Sci U S A 105, 19474-19479.
Kos, B., Suskovic, J., Vukovic, S., Simpraga, M., Frece, J., Matosic, S.,2003, Adhesion and aggregation ability of probiotic strain Lactobacillus acidophilus M92. J Appl Microbiol 94,981-987.
Kumar, A., Wu, H., Collier-Hyams, L.S., Hansen, J.M., Li, T., Yamoah, K., Pan, Z.Q., Jones, D.P., Neish, A.S.,2007, Commensal bacteria modulate cullin-dependent signaling via generation of reactive oxygen species. EMBO J 26,4457-4466.
Lee, J.H., Halgerson, J.S., Kim, J.H., O'Sullivan, D.J.,2007, Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector. Appl Environ Microbiol 73,4417-4424.
Leer, R.J., van Luijk, N., Posno, M., Pouwels, P.H.,1992, Structural and functional analysis of two cryptic plasmids from Lactobacillus pentosus MD353 and Lactobacillus plantarum ATCC 8014. Mol Gen Genet 234,265-274.
Lilly, D.M., Stillwell, R.H.,1965, Probiotics:Growth-Promoting Factors Produced by Microorganisms. Science 147,747-748.
Lin, P.W., Myers, L.E., Ray, L., Song, S.C., Nasr, T.R., Berardinelli, A.J., Kundu, K., Murthy, N., Hansen, J.M., Neish, A.S.,2009, Lactobacillus rhamnosus blocks inflammatory signaling in vivo via reactive oxygen species generation. Free Radic Biol Med 47,1205-1211.
Liu, Y.G., Whittier, R.F.,1995, Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25,674-681.
Llopis, M., Antolin, M., Carol, M., Borruel, N., Casellas, F., Martinez, C., Espin-Basany, E., Guarner, F., Malagelada, J.R.,2009, Lactobacillus casei downregulates commensals'inflammatory signals in Crohn's disease mucosa. Inflamm Bowel Dis 15,275-283.
Lokman, B.C., van Santen, P., Verdoes, J.C., Kruse, J., Leer, R.J., Posno, M., Pouwels, P.H.,1991, Organization and characterization of three genes involved in D-xylose catabolism in Lactobacillus pentosus. Mol Gen Genet 230,161-169.
Ma, D., Forsythe, P., Bienenstock, J.,2004, Live Lactobacillus rhamnosus is essential for the inhibitory effect on tumor necrosis factor alpha-induced interleukin-8 expression. Infect Immun 72,5308-5314.
Mack, D.R., Ahrne, S., Hyde, L., Wei, S., Hollingsworth, M.A.,2003, Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut 52,827-833.
Marraffini, L.A., Dedent, A.C., Schneewind, O.,2006, Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70,192-221.
Mierau, I., Kleerebezem, M.,2005,10 years of the nisin-controlled gene expression system (NICE) in Lactococcus lactis. Appl Microbiol Biotechnol 68,705-717.
Narita, J., Ishida, S., Okano, K., Kimura, S., Fukuda, H., Kondo, A.,2006, Improvement of protein production in lactic acid bacteria using 5'-untranslated leader sequence of slpA from Lactobacillus acidophilus. Appl Microbiol Biotechnol 73,366-373.
Neish, A.S., Gewirtz, A.T., Zeng, H., Young, A.N., Hobert, M.E., Karmali, V., Rao, A.S., Madara, J.L.,2000, Prokaryotic regulation of epithelial responses by inhibition of IkappaB-alpha ubiquitination. Science 289,1560-1563.
O'Hara, A.M., O'Regan, P., Fanning, A., O'Mahony, C., Macsharry, J., Lyons, A., Bienenstock, J., O'Mahony, L., Shanahan, F.,2006, Functional modulation of human intestinal epithelial cell responses by Bifidobacterium infantis and Lactobacillus salivarius. Immunology 118,202-215.
Oozeer, R., Furet, J.P., Goupil-Feuillerat, N., Anba, J., Mengaud, J., Corthier, G., 2005, Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice. Appl Environ Microbiol 71,1356-1363.
Oozeer, R., Goupil-Feuillerat, N., Alpert, C.A., van de Guchte, M., Anba, J., Mengaud, J., Corthier, G.,2002, Lactobacillus casei is able to survive and initiate protein synthesis during its transit in the digestive tract of human flora-associated mice. Appl Environ Microbiol 68,3570-3574.
Ouwehand, A.C., Tuomola, E.M., Tolkko, S., Salminen, S.,2001, Assessment of adhesion properties of novel probiotic strains to human intestinal mucus. Int J Food Microbiol 64,119-126.
Pelletier, C., Bouley, C., Cayuela, C., Bouttier, S., Bourlioux, P., Bellon-Fontaine, M.N.,1997, Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains. Appl Environ Microbiol 63,1725-1731.
Perez-Arellano, I., Perez-Martinez, G.,2003, Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei. FEMS Microbiol Lett 222,123-127.
Petrof, E.O., Claud, E.C., Sun, J., Abramova, T., Guo, Y., Waypa, T.S., He, S.M., Nakagawa, Y., Chang, E.B.,2009, Bacteria-free solution derived from Lactobacillus plantarum inhibits multiple NF-kappaB pathways and inhibits proteasome function. Inflamm Bowel Dis 15,1537-1547.
Pouwels, P.H., Leer, R.J.,1993, Genetics of lactobacilli:plasmids and gene expression. Antonie Van Leeuwenhoek 64,85-107.
Pouwels, P.H., van Luijk, N., Leer, R.J., Posno, M.,1994, Control of replication of the Lactobacillus pentosus plasmid p353-2:evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein. Mol Gen Genet 242,614-622.
Pretzer, G., Snel, J., Molenaar, D., Wiersma, A., Bron, P.A., Lambert, J., de Vos, W.M., van der Meer, R., Smits, M.A., Kleerebezem, M.,2005, Biodiversity-based identification and functional characterization of the mannose-specific adhesin of Lactobacillus plantarum. J Bacteriol 187, 6128-6136.
Reid, G., Burton, J.,2002, Use of Lactobacillus to prevent infection by pathogenic bacteria. Microbes Infect 4,319-324.
Resta-Lenert, S., Barrett, K.E.,2003, Live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive Escherichia coli (EIEC). Gut 52,988-997.
Resta-Lenert, S., Barrett, K.E.,2006, Probiotics and commensals reverse TNF-alpha-and IFN-gamma-induced dysfunction in human intestinal epithelial cells. Gastroenterology 130,731-746.
Rinkinen, M., Westermarck, E., Salminen, S., Ouwehand, A.C.,2003, Absence of host specificity for in vitro adhesion of probiotic lactic acid bacteria to intestinal mucus. Vet Microbiol 97,55-61.
Rojas, M., Ascencio, F., Conway, P.L.,2002, Purification and characterization of a surface protein from Lactobacillus fermentum 104R that binds to porcine small intestinal mucus and gastric mucin. Appl Environ Microbiol 68, 2330-2336.
Ruiz, P.A., Hoffmann, M., Szcesny, S., Blaut, M., Haller, D.,2005, Innate mechanisms for Bifidobacterium lactis to activate transient pro-inflammatory host responses in intestinal epithelial cells after the colonization of germ-free rats. Immunology 115,441-450.
Saarela, M., Mogensen, G., Fonden, R., Matto, J., Mattila-Sandholm, T.,2000, Probiotic bacteria:safety, functional and technological properties. J Biotechnol 84,197-215.
Schlee, M., Harder, J., Koten, B., Stange, E.F., Wehkamp, J., Fellermann, K.,2008, Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp Immunol 151,528-535.
Schmieger, H., Schicklmaier, P.,1999, Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104. FEMS Microbiol Lett 170, 251-256.
Seth, A., Yan, F., Polk, D.B., Rao, R.K.,2008, Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC-and MAP kinase-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 294, G1060-1069.
Sillanpaa, J., Martinez, B., Antikainen, J., Toba, T., Kalkkinen, N., Tankka, S., Lounatmaa, K., Keranen, J., Hook, M., Westerlund-Wikstrom, B., Pouwels, P.H., Korhonen, T.K.,2000, Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus. J Bacteriol 182,6440-6450.
Smit, E., Jager, D., Martinez, B., Tielen, F.J., Pouwels, P.H.,2002, Structural and functional analysis of the S-layer protein crystallisation domain of Lactobacillus acidophilus ATCC 4356:evidence for protein-protein interaction of two subdomains. J Mol Biol 324,953-964.
Smit, E., Oling, F., Demel, R., Martinez, B., Pouwels, P.H.,2001, The S-layer protein of Lactobacillus acidophilus ATCC 4356:identification and characterisation of domains responsible for S-protein assembly and cell wall binding. J Mol Biol 305,245-257.
Smit, E., Pouwels, P.H.,2002, One repeat of the cell wall binding domain is sufficient for anchoring the Lactobacillus acidophilus surface layer protein. J Bacteriol 184,4617-4619.
Sorvig, E., Skaugen, M., Naterstad, K., Eijsink, V.G., Axelsson, L.,2005b, Plasmid p256 from Lactobacillus plantarum represents a new type of replicon in lactic acid bacteria, and contains a toxin-antitoxin-like plasmid maintenance system. Microbiology 151,421-431.
Tao, Y., Drabik, K.A., Waypa, T.S., Musch, M.W., Alverdy, J.C., Schneewind, O., Chang, E.B., Petrof, E.O.,2006, Soluble factors from Lactobacillus GG activate MAPKs and induce cytoprotective heat shock proteins in intestinal epithelial cells. Am J Physiol Cell Physiol 290, C1018-1030.
Tassell, M.L., Miller, M.J.,2011, Lactobacillus adhesion to mucus. Nutrients 3, 613-636.
Tien, M.T., Girardin, S.E., Regnault, B., Le Bourhis, L., Dillies, M.A., Coppee, J.Y., Bourdet-Sicard, R., Sansonetti, P.J., Pedron, T.,2006, Anti-inflammatory effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells. J Immunol 176,1228-1237.
Tiurin, M.V.,1992, [Electrotransformation of bacteria]. Antibiot Khimioter 37,51-55.
Tuomola, E., Crittenden, R., Playne, M., Isolauri, E., Salminen, S.,2001, Quality assurance criteria for probiotic bacteria. Am J Clin Nutr 73,393S-398S.
Vadillo-Rodriguez, V., Busscher, H.J., Norde, W., de Vries, J., van der Mei, H.C., 2004, Dynamic cell surface hydrophobicity of Lactobacillus strains with and without surface layer proteins. J Bacteriol 186,6647-6650.
van de Guchte, M., Penaud, S., Grimaldi, C., Barbe, V., Bryson, K., Nicolas, P., Robert, C., Oztas, S., Mangenot, S., Couloux, A., Loux, V., Dervyn, R., Bossy, R., Bolotin, A., Batto, J.M., Walunas, T., Gibrat, J.F., Bessieres, P., Weissenbach, J., Ehrlich, S.D., Maguin, E.,2006, The complete genome sequence of Lactobacillus bulgaricus reveals extensive and ongoing reductive evolution. Proc Natl Acad Sci U S A 103,9274-9279.
van Pijkeren, J.P., Canchaya, C., Ryan, K.A., Li, Y., Claesson, M.J., Sheil, B., Steidler, L., O'Mahony, L., Fitzgerald, G.F., van Sinderen, D., O'Toole, P.W., 2006, Comparative and functional analysis of sortase-dependent proteins in the predicted secretome of Lactobacillus salivarius UCC118. Appl Environ Microbiol 72,4143-4153.
Velez, M.P., De Keersmaecker, S.C., Vanderleyden, J.,2007, Adherence factors of Lactobacillus in the human gastrointestinal tract. FEMS Microbiol Lett 276, 140-148.
Ventura, M., Jankovic, I., Walker, D.C., Pridmore, R.D., Zink, R.,2002, Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri. Appl Environ Microbiol 68,6172-6181.
Vidgren, G., Palva, I., Pakkanen, R., Lounatmaa, K., Palva, A.,1992, S-layer protein gene of Lactobacillus brevis:cloning by polymerase chain reaction and determination of the nucleotide sequence. J Bacteriol 174,7419-7427.
Voltan, S., Martines, D., Elli, M., Brun, P., Longo, S., Porzionato, A., Macchi, V., D'Inca, R., Scarpa, M., Palu, G., Sturniolo, G.C., Morelli, L., Castagliuolo, I., 2008, Lactobacillus crispatus M247-derived H2O2 acts as a signal transducing molecule activating peroxisome proliferator activated receptor-gamma in the intestinal mucosa. Gastroenterology 135,1216-1227.
Vujcic, M., Topisirovic, L.,1993, Molecular analysis of the rolling-circle replicating plasmid pAl of Lactobacillus plantarum A112. Appl Environ Microbiol 59, 274-280.
Wu, G.D., Huang, N., Wen, X., Keilbaugh, S.A., Yang, H.,1999, High-level expression of I kappa B-beta in the surface epithelium of the colon:in vitro evidence for an immunomodulatory role. J Leukoc Biol 66,1049-1056.
Zhang, L., Li, N., Caicedo, R., Neu, J.,2005, Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factor-alpha-induced interleukin-8 production in Caco-2 cells. J Nutr 135,1752-1756.
Zhang, L., Xu, Y.Q., Liu, H.Y., Lai, T., Ma, J.L., Wang, J.F., Zhu, Y.H.,2010, Evaluation of Lactobacillus rhamnosus GG using an Escherichia coli K88 model of piglet diarrhoea:Effects on diarrhoea incidence, faecal microflora and immune responses. Vet Microbiol 141,142-148.
蔡凯凯,黄占旺,叶德军,孙长涛.益生菌调节肠道菌群及免疫调节作用机理[J].中国饲料,2011,18：34-37.
刘延国,张琳,冯香安,李杰.益生菌在消化道中的抑菌作用[J].中国饲料,2011,16：9-12.
罗丽娟,施季森.一种DNA侧翼序列分离技术——TAIL-PCR[J].南京林业大学学报,2003,27：87-90.