单核细胞增生李斯特菌毒力基因inlB/actA双缺失突变株的构建

单核细胞增生李斯特菌inlB和actA毒力基因的编码产物InlB和ActA是与其致病性相关的重要毒力因子,与该菌在细胞间扩散、传播有着直接的关系,其中肌动蛋白聚集因子ActA是细菌由细胞浆扩散至相邻细胞所必须的因子,而内化素InlB在对肝细胞的侵袭过程中起着重要作用。本研究中利用同源重组技术在inlB缺失菌株基础上成功构建了毒力基因inlB和actA双缺失的突变株,获得减毒突变株,为构建预防人类和动物疾病的疫苗载体奠定了基础。     

InlA和InlB介导单核细胞增生李斯特菌入侵宿主细胞分子机制的研究进展

单核细胞增生李斯特菌(Listeria monocytogenes)是一种革兰氏阳性食源性致病菌。在造成宿主食源性感染的过程中, 单核细胞增生李斯特菌能凭借其独特的表面蛋白入侵宿主的非吞噬细胞。内化素蛋白家族(Internalins)是介导单核细胞增生李斯特菌入侵宿主非吞噬细胞的主要因子。本文根据国内外一些最新的研究成果, 结合作者近几年的工作, 综述了在侵染宿主的过程中, 单核细胞增生李斯特菌主要的内化素蛋白InlA和InlB介导细菌入侵宿主细胞的分子机制, 以期为阐明食源性致病菌致病机理、预防和治疗食源性疾病提供理论基础。     

一株ST477型单增李斯特菌的全基因组测序及inlA基因遗传多样性

【背景】单增李斯特菌是一种重要的条件致病菌,不同型别菌株在宿主范围和毒力等方面存在差异。内化素基因inlA在入侵宿主上皮细胞中具有重要作用。【目的】研究单增李斯特菌序列型(Sequence type,ST)为477菌株的基因组特征及内化素基因inlA的遗传多样性。【方法】使用相关软件对测序数据进行多位点序列分型(Mutilocussequencetyping,MLST)、单核苷酸多态性(Single nucleotide polymorphism,SNP)及基因inlA遗传多样性分析。【结果】MLST进化分析结果显示,分离自不同国家的菌株具有较近亲缘关系。以分离自中国食品的ST477型菌株为参考菌株,通过SNP分析表明,加拿大食品中的ST9型菌株发生的突变位点最少(91-93个)。7株复合克隆系(Clonal complex,CC)为9的菌株其inlA基因序列间核苷酸相似性为29.8%-100%。【结论】初步分析了ST477型别菌株的进化及基因组特征,同时研究了部分CC9克隆系菌株inlA基因突变情况,为研究ST477型别菌株的进化及单增李斯特菌的毒力提供基础数据。     

天然缺失inlAB的非典型单核细胞增多性李斯特菌生物学特性鉴定

摘要：【目的】 InlA与InlB是单核细胞增多性李斯特菌重要的毒力因子,其介导的黏附作用是细菌建立感染的前提。本研究拟探明天然缺失inlAB基因簇的非典型单增李斯特菌的表型与基因型特征。【方法】针对inlAB天然缺失株S10,进行生化特征、细胞黏附力、小鼠体内毒力、感染相关基因检测、谱系分析等。【结果】 S10株为具有典型单增李斯特菌生化特征的1/2b型菌株,对HeLa细胞的黏附力显著低于其他菌株（p<0.05）,对小鼠毒力较弱。S10缺失inlAB及与其毗邻的lmo0431、lmo0432、lmo0436、lmo0437基因,但具有李斯特菌第一毒力岛中完整的毒力基因构成。S10分布于谱系Ⅰ的进化枝上,与4b型菌株的遗传距离较近。【结论】 S10为单增李斯特菌inlAB天然缺失株代表该类非典型菌株的首次报道。S10具有典型的单增李斯特菌谱系Ⅰ基因背景,inlAB可能通过独立的重组或水平转移事件缺失于基因组。     

基因dat对单核细胞增生李斯特氏菌的生物学特性影响

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是一种重要的食源性致病菌,其基因dat编码D-丙氨酸氨基转移酶,可将D-谷氨酸转变为D-丙氨酸,后者是细菌细胞壁肽聚糖的组成成分。在Lm野生株EGDe actA及inlB双基因缺失株(EGDeΔactAΔinlB)的基础上,利用同源重组的方法进一步构建了缺失基因dat的菌株(EGDeΔactAΔinlBΔdat),并对该缺失菌株生长状态、毒力基因表达水平、细胞侵袭及生物被膜的形成量等基本生物学特性进行研究,运用Real Time-PCR(RT-PCR)测定Lm毒力基因的相对表达量。结果表明,基因的缺失对突变菌株的生长能力以及生物被膜形成有重要的调控作用,并导致毒力基因srt A、plc A表达量下调,VIP、inlA表达量上调,对Coca-2细胞的侵袭无影响。此缺失株的构建为进一步研究基因dat的功能提供了材料。     

clpE基因缺失对肺炎链球菌毒力的影响

【目的】 探索clpE基因缺失对肺炎链球菌毒力的影响。【方法】 用长臂同源多聚酶链式反应(LFH-PCR)方法失活clpE基因,用PCR、测序鉴定缺失菌株,通过动物实验观察clpE基因缺失株毒力改变情况, 同时用细胞实验比较clpE基因缺失株和野生菌对宿主细胞的粘附和侵袭能力,最后用实时荧光定量PCR分析自溶素(major autolysin A,lytA)、表面黏附素A(pneumococcal surface adhesion A,psaA)、溶血素(pneumolysin,ply)、肺炎球菌表面蛋白A（pneumococcal surface protein A, pspA)和神经氨酸酶（neuraminidase, nanA）的表达。 【结果】小鼠毒力实验表明野生菌株半数致死时间54h,而缺失株半数致死时间为21d,两者比较有统计学差异（P<0 .0l）;缺失菌在对宿主细胞的粘附能力明显低于野生菌株（P<0.05）。实时荧光定量PCR显示clpE缺失株的五个毒力因子mRNA表达水平均低于野生菌,两者比较有统计学差异（P<0. 05）;【结论】ClpE通过调控肺炎链球菌多种毒力因子表达,而影响其毒力。     

单核细胞增生李斯特菌PrfA蛋白转录调控毒力基因表达的分子机制

单核细胞增生李斯特菌 (Listeria monocytogenes LM) 属于典型的细胞内寄生革兰氏阳性菌, 是WHO公布的四大食源性致病菌之一。LM不仅是人畜共患传染病李斯特菌病 (listeriosis) 的主要病原菌, 也是研究胞内感染和细胞介导的免疫应答的模式细菌。绝大多数LM毒力基因的转录表达受到PrfA蛋白的调控。本文简单介绍了LM侵染宿主细胞必需的毒力基因及其产物; 重点对毒力基因调节蛋白PrfA的结构和功能, PrfA调节毒力基因表达的主要方式最新进展进行了综述和讨论。     

单核细胞增生李斯特菌PrfA蛋白转录调控毒力基因表达的分子机制

单核细胞增生李斯特菌(Listeria monocytogenes LM)属于典型的细胞内寄生革兰氏阳性菌,是WHO公布的四大食源性致病菌之一.LM不仅是人畜共患传染病李斯特菌病(listeriosis)的主要病原菌,也是研究胞内感染和细胞介导的免疫应答的模式细菌.绝大多数LM毒力基因的转录表达受到PrfA蛋白的调控.本文简单介绍了LM侵染宿主细胞必需的毒力基因及其产物;重点对毒力基因调节蛋白PrfA的结构和功能,PrfA调节毒力基因表达的主要方式最新进展进行了综述和讨论.     

透明质酸生产菌溶血素S基因缺失突变菌株的构建及其特性北大核心CSCD

【目的】马链球菌兽疫亚种是工业上生产透明质酸的主要菌种,该菌能产生引起宿主细胞溶血的链球菌溶血素S(streptolysin S,SLS)毒素,因而其产品的安全性一直是人们所担心的问题。本实验的目的就是通过基因敲除的方法构建不产SLS的透明质酸生产工程菌,同时探讨溶血素sag A基因缺失对菌株透明质酸合成和其他毒力因子的影响。【方法】利用温度敏感/自杀性质粒p JR700载体系统,构建马链球菌兽疫亚种sag A基因缺失突变株;通过PCR扩增,溶血平板和SLS含量测定等方法确定sag A基因缺失;采用分光光度、SDS-PAGE和细胞毒性试验等分析方法,对野生菌株和sag A基因缺失突变菌株透明质酸含量、透明质酸分子量、溶血素Hylc、透明质酸分解酶、甘油醛-3-磷酸脱氢酶和菌体表面蛋白等相关毒力因子进行对比研究。【结果】获得了透明质酸产量提高30%而溶血活性极低的马链球菌兽疫亚种sag A基因缺失突变株。该突变株与野生菌株相比较,透明质酸分解酶活性增加而透明质酸相对分子量降低,此外,与毒力相关的表面蛋白含量、溶血素Hylc和甘油醛-3-磷酸脱氢酶活性也显著降低。细胞毒性实验结果表明,野生菌株与sag A基因缺失突变菌株的培养物上清液,对细胞活性的影响存在显著差异。【结论】在马链球菌兽疫亚种中sag A不仅是表达溶血素SLS的基因,同时sag A基因对菌株透明质酸合成、透明质酸分解酶、菌体表面蛋白、溶血素Hylc和甘油醛-3-磷酸脱氢酶等都具有调节作用。     

RsbV基因缺失对单核细胞增生李斯特菌毒力的影响

[目的]探讨RsbV基因缺失对单核细胞增生李斯特菌(LM)毒力的影响.[方法]运用基因重叠延伸PCR( SOE-PCR)技术扩增出LM-XS5野毒株的RsbV基因缺失片段,然后用同源重组方法构建RsbV基因缺失株;通过肝脾细菌计数、LD50的测定和毒力基因转录水平的检测(qRT-PCR),研究LM野毒株和缺失株在毒力上的差异.[结果]RsbV基因缺失株LD50是野毒株的104倍(P＜0.01);缺失株在小白鼠肝和脾内的载菌量均明显减少(P＜0.05);实时荧光定量PCR检测结果发现,缺失株4个毒力因子的表达水平均显著低于野毒株(P＜0.05);缺失株免疫小白鼠后对野毒株的攻毒具有良好的免疫保护作用.[结论]RsbV对LM的4个毒力基因inlA、LLO、PlcA和PrfA的表达具有调控作用;RsbV基因缺失株毒力明显减弱,但仍保留了较强的免疫原性.     

A Common Clathrin‐Mediated Machinery Co‐ordinates Cell–Cell Adhesion and Bacterial Internalization

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E‐cadherin, hijack the host adherens junction (AJ) machinery and invade non‐phagocytic cells by a clathrin‐dependent mechanism. Here, we investigate a potential role for clathrin in cell–cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell–cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E‐cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E‐cadherin. We demonstrate that non‐adherent cells expressing the InlA chimera, as bacteria, can be internalized by E‐cadherin‐expressing adherent cells. Together these results reveal that a common clathrin‐mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.     

The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice

In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD. These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins. The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-β-glucosidase and the succinyl-diaminopimelate desuccinylase of E. coli. A similar internalin gene cluster, inlC2DE, localised to the same position on the L. monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615–1625). Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3′-terminal end of inlC2 and the 5′-terminal part of inlD. The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene. Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA- independent promoter located upstream of inlG. PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L. monocytogenes isolates tested. A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain. Received: 21 April 1998 / Accepted: 5 June 1998     

Bacterial entry into cells: a role for the endocytic machinery

Increasing evidence indicates that pathogens have evolved highly efficient strategies to induce their internalization within host cells. Viruses and bacteria express and expose on their surface, molecules that mimic endogenous ligands to cell receptors, thereby inducing specific intracellular signalling cascades. More recently it has become clear that, as most viruses, bacteria can enter cells via the clathrin-mediated pathway, indicating a key role for endocytosis in pathogens entry into cells. Here we review the pathways followed by Listeria monocytogenes to enter into non-phagocytic cells, as a model for the subversion of cellular functions to induce pathogens internalization.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Non-specificity of a colorimetric method for the estimation of N-acetyl-d-glucosamine

The colorimetric method of Reissig et al. for the estimation of N-acetylamino sugars, is often used as a specific method for the quantification of the N-acetyl-d-glucosamine. Although this assay is more sensitive to the monomer, it recognizes all soluble N-acetyl-d-glucosamine oligomers. This result is very important because this method is extensively used in biology for the estimation of chitinolytic activity.     

Listerial invasion protein internalin B promotes entry into ileal Peyer's patches in vivo

Listeria monocytogenes (Lm) invades the host intestine using listerial invasion proteins, internalins. The in vivo role of internalin A (InlA) and internalin B (InlB) is reported here. Intragastric (i.g.) administration and ligated loop assays with ΔinlB-Lm demonstrated that a lack of InlB significantly attenuates the invasive ability of Lm into various organs. On the other hand, InlA(m)-Lm expressing a mutant InlA with two substitutions, S192N and Y369S, which has been reported to increase the affinity of InlA to mouse E-cadherin, resulted in little increase in intestinal infection according to both ligated loop and i.g. infection assays. Lm preferentially enters ileal Peyer's patch (PP) via M cells and ΔinlB-Lm showed severely reduced ability to invade though these cells. The present results reveal the importance of InlB, which accelerates listerial invasion into M cells on ileal PPs in vivo.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.