单增李斯特菌耐药外排泵研究进展

单增李斯特菌是一种重要的人兽共患食源性胞内致病菌,广泛存在于自然环境中且易污染动物性食品,人及动物感染后可引起严重的李斯特菌病,死亡率高达30%。单增李斯特菌通常对多种药物敏感,然而,因不合理使用抗菌药或消毒剂形成的选择压力导致李斯特菌多重耐药情况的报道日渐增多。外排泵蛋白是细菌中一类重要的蛋白,可参与机体多种生物学过程,包括影响细菌对抗生素敏感性、促进有毒化合物泵出、影响细菌毒力等。本文综述了近年来关于单增李斯特菌耐药外排泵的功能及调控机制的研究进展,为深入理解李斯特菌耐药等环境适应机制及有效控制该病原污染传播和筛选抗感染药物新靶点提供理论基础。     

单增李斯特菌溶血素O的功能研究进展

单核细胞增生李斯特菌(Listeria monocytogenes, Lm,简称单增李斯特菌)是一种普遍存在的革兰阳性食源性病原体,可引起人类和一些动物的李斯特菌病。侵袭性李斯特菌病通常很严重,临床上表现为自然流产、败血症和脑膜脑炎,也可表现为发热性胃肠炎综合症。成孔蛋白单增李斯特菌溶血素O(Listeriolysin O,LLO,由hly基因编码)是一种重要的毒力因子,属于胆固醇依赖性细胞溶解素(cholesterol-dependent cytolysins,CDC)毒素,其通过膜穿孔机制介导Lm从吞噬体逃逸并引起李斯特菌病。最近的研究表明LLO除了主要的膜穿孔作用,还存在其他功能,在Lm感染过程中扮演了重要的角色。从LLO的功能和作用机制等方面综述了近些年对该毒素的研究进展,以便更好地理解单增李斯特菌的感染机制,为防治李斯特病的相关研究提供参考。     

单增李斯特菌LPXTG蛋白Lmo0880在感染致病中的作用

【目的】通过构建单核细胞增生李斯特菌(单增李斯特菌) LPXTG蛋白Lmo0880的基因缺失菌株和回补菌株,探究Lmo0880在细菌生长、细胞感染和宿主感染等方面发挥的作用。【方法】利用同源重组原理构建lmo0880的基因缺失株及回补株,比较野生株、缺失株和回补株在生长能力、细胞黏附与侵袭和胞内增殖能力等方面的差异,从而鉴定Lmo0880在单增李斯特菌感染宿主中的作用。【结果】缺失lmo0880基因后,单增李斯特菌在生长能力上无明显变化;对细胞的黏附能力无显著差异,但对细胞侵袭能力、胞内增殖能力、小鼠致病力和小鼠组织定殖能力显著降低。【结论】本研究阐明了单增李斯特菌LPXTG蛋白Lmo0880在细胞侵袭、胞内增殖和组织定殖等方面发挥的重要作用。     

单增李斯特菌氧化还原蛋白系统研究进展

单增李斯特菌是重要的食源性病原微生物,抗氧化应激是李斯特菌生存和致病的关键机制之一。活性氧(reactive oxygen species,ROS)浓度升高会破坏氧化还原平衡,使机体处于氧化胁迫的应激状态,进而导致生物大分子如蛋白质的损伤。蛋白质中半胱氨酸等含硫氨基酸对ROS尤其敏感,半胱氨酸残基脱氢氧化生成二硫键,可以稳定蛋白质空间构象,增加蛋白质的半衰期,进而使蛋白质免受损坏。抗氧化修复通常指的是对半胱氨酸残基的氧化还原过程,即二硫键的形成与打开。硫氧还蛋白家族包含硫氧还蛋白、谷氧还蛋白和Dsb-样蛋白系统,是生物体中常见的氧化还原修复系统。本文根据现有的文献报道,结合本课题组的研究进展,对单增李斯特菌硫氧还蛋白家族进行综述,以期为完善单增李斯特菌硫氧还蛋白调控系统提供参考。     

单增李斯特菌lmo2192基因克隆与表达

[目的]对单增李斯特菌新疆绵羊脑炎临床分离株LM90SB2的lmo2192基因进行克隆及原核表达。[方法]利用PCR方法扩增lmo2192基因,连接p MD19-T载体进行克隆,筛选阳性菌进行测序比对。构建重组表达质粒p ET32a-2192,将其转入大肠杆菌感受态细胞,经诱导表达,利用SDS-PAGE与Western Blotting鉴定重组蛋白。[结果]扩增lmo2192基因序列长度为969 bp,与预期一致。该基因在大肠杆菌中大量表达,经SDS-PAGE和Western Blotting鉴定分析该产物为56 k Da左右的融合重组蛋白,与预期大小一致。[结论]成功克隆lmo2192基因,并获得大量表达,为进一步研究该基因功能奠定理论基础。     

单增李斯特菌在冷鲜猪肉中的生长预测模型比较

[目的]探讨单增李斯特菌(Listeria monocytogenes,LM)在冷鲜猪肉中的生长及现有预测软件GP( Growth Predictor)、PMP( Pathogen Modeling Programme)及CP (ComBase Predictor)对LM在冷鲜猪肉中生长的预测准确性.[方法]测定LM在4℃、8℃、12℃及16℃冷鲜猪肉中的生长,并用DMFit软件对生长数据进行拟合,计算各个温度点下的迟滞期(Lag phase duration,LPD)、生长率(Growth rate,GR)、最大生长密度(Maximum population density,MPD),同时用3种预测模型对相同条件下LM在猪肉中的生长进行预测,将实测值与预测值进行比较分析.[结果]冷鲜猪肉在16℃,经过2.6h LM即进入对数生长期.从8℃提高至12℃时,LM在冷鲜猪肉中的生长率从0.017l0g(cfu/g).h-1增至0.038l0g(cfu/g).h-1.在4℃ - 16℃,PMP预测的GR要比实测值低,而LPD则高于实测值.GP在8℃及以上的温度范围内,所预测LPD比实测值偏高.3种预测模型中,GP对GR的预测稍高于实测值,偏差因子(Bf)为1.01,准确因子(Af)为1.38;CP对LPD的预测值与实测值更为接近,Af及Bf分别为4.33及2.83.[结论]在冷鲜猪肉生产和销售过程中,严格控制温度尤为重要.PMP的预测较为保守,不适于冷鲜猪肉中LM生长的预测;建议用GP对GR进行预测,而CP对LPD的预测仅作为参考.     

单增李斯特菌生物膜及其形成机制的研究进展

单增李斯特菌(Lm)是重要的人兽共患食源性病原菌。Lm生物膜与其致病性和耐药性密切相关。影响Lm生物膜形成的关键因子有鞭毛糖蛋白、胞外基质和群体感应系统等。鞭毛糖蛋白能促进菌体聚集,从而直接影响生物膜的形成。胞外DNA参与Lm粘附和生物膜早期的形成,并与胞外多糖和胞外结合蛋白一起构成生物膜胞外基质。Lm的Agr群体感应系统正调控生物膜形成,是一种集合毒力因子、耐药因子和生物膜的整体水平调控网络体系。     

单增李斯特菌胎盘感染的体内外模型研究进展

单核细胞增生李斯特氏菌（Listeria monocytogenes）是重要的食源性致病菌,能引发人类的李斯特菌病,是全球公共卫生问题之一。该菌易感染孕妇,引起胎儿和新生儿的侵袭性李斯特菌病,严重威胁母婴健康。因此,建立有效的单增李斯特菌感染胎盘体内外模型,解析和探究单增李斯特菌经胎盘感染机制,是预防和控制单增李斯特菌感染母婴的关键所在。本文综述了可用于研究单增李斯特菌母婴感染的体内外胎盘模型,总结和讨论了各类模型的优势和局限性;并着重分析了体外三维胎盘屏障模型在单增李斯特菌感染方面的研究进展和未来研究方向。以期为深入解析该菌经胎盘感染的途径、发病机制提供支持,并为预防和控制母婴李斯特菌病提供科学参考。     

单增李斯特菌末端氧化酶亚基QoxB的生物学功能探究

【目的】本研究旨在构建单增李斯特菌末端细胞色素aa3氧化酶亚基qoxB基因缺失株,并探索其在细菌生长及感染过程中发挥的生物学功能。【方法】利用同源重组方法构建获得缺失株ΔqoxB后,对野生株EGD-e和缺失株ΔqoxB的生长能力、细菌运动能力和细胞内黏附、侵袭、增殖及胞内迁移能力进行比较,同时利用荧光定量PCR方法检测ΔqoxB中鞭毛相关基因转录水平的变化。【结果】缺失qoxB基因后细菌在体外培养过程中生长能力没有差异,细菌的鞭毛运动能力显著降低,在30℃培养24 h和48 h后ΔqoxB运动圈直径分别较EGD-e下降35.86%和34.20%,且22个鞭毛相关基因转录水平显著降低。通过细胞感染试验发现缺失qoxB基因后细胞黏附、侵袭、增殖及胞内迁移能力均显著下降。【结论】本研究首次证实末端氧化酶亚基QoxB能降低单增李斯特菌的运动能力和对细胞的感染能力,此研究为进一步阐明末端细胞色素氧化酶影响单增李斯特菌的致病机制提供重要依据。     

超高压对单增李斯特菌细胞膜的损伤和致死机理

【目的】研究超高压对病原微生物单增李斯特菌细胞膜损伤的影响。【方法】本文以单增李斯特菌为研究对象,探讨了不同压力处理(100-500 MPa)对单增李斯特菌的灭活作用,利用透射电镜观察高压处理对细菌细胞超微结构的影响,通过紫外分光光度法、原子吸收分光光度法和荧光分光光度法测定高压处理对细菌细胞膜通透性的影响,采用超微量Na+/K+-ATP酶试剂盒测定高压处理对细菌细胞膜Na+/K+-ATP酶活力的影响。【结果】25℃经300、350、400 MPa压力处理15 min后,单增李斯特菌总数由9.00分别降至5.20、3.27、1.35个对数单位,经450MPa及以上的压力处理后,单增李斯特菌的致死率达到100%。超高压处理对单增李斯特菌的细胞超微结构造成明显的损伤,细胞结构不完整,细胞壁局部被破坏,细胞膜通透性增大,细胞内物质聚合,出现透电子区。由于细胞膜的损伤使得细胞内无机盐离子、紫外吸收物质流出,细胞膜上的Na+/K+-ATPase失活。【结论】超高压处理造成单增李斯特菌细胞形态结构明显损伤,改变细胞膜的通透性,降低细胞膜上Na+/K+-ATP酶活力,最终使得细胞内无机盐离子和胞内大分子物质外流而死亡。     

Surface Listeria monocytogenes carbohydrate-binding components revealed by agglutination with neoglycoproteins

Carbohydrate-binding components were shown to be present at the surface of Listeria monocytogenes by means of a panel of neoglycoproteins using direct agglutination. These lectin-like components bind on neoglycoproteins bearing d -glucosamine, l -fucosylamine, or para-amino-phenyl-α- d -mannopyrannoside residues. The interactions were inhibited by the carbohydrate moieties specific to the neoglycoproteins. The protein nature of the lectin-like components of L. monocytogenes was ascertained by the loss of carbohydratebinding capacity following protease treatment.     

A multidrug efflux transporter in Listeria monocytogenes

A chromosomal gene (mdrL) was found in Listeria monocytogenes L028, showing a high degree of similarity with multidrug efflux transporters of the major facilitator superfamily (family 2). An allele-substituted mutant of this gene failed to pump out ethidium bromide and presented lower minimal inhibitory concentrations of macrolides, cefotaxime and heavy metals. This is the first multidrug efflux pump described in Listeria.     

李斯特菌生物被膜形成的调控

单核细胞增生李斯特菌(Listeria monocyiogenes)能引起人和动物脑膜炎、败血症、流产和单核细胞增多等症状,临床发病率在美国和欧洲等西方发达国家大约为2-8例/10万人,死亡率20％-30％或更高,被WHO列为关系食品卫生安全的重要病源细菌之一一[1-2].该菌能在多数固体表面形成生物被膜,在食品生产、加工、运输和保藏过程中,一旦发生细菌感染并形成生物被膜便难以将其彻底清除,严重威胁着食品卫生安全[3],但其生物被膜形成的具体分子机制尚不清楚[4].     

Fibronectin‐binding protein,FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA‐deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild‐type L. monocytogenes. FbpA did not affect the amounts of various virulence‐determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild‐type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.     

Utilization of transferrin-bound iron by Listeria monocytogenes

Abstract It has been demonstrated that under iron-restricted conditions, Listeria monocytogenes can utilize iron-loaded transferrin (Tf) from a range of species as its sole source of iron for growth. Human transferrin conjugated to horseradish-peroxidase (HRP-Tf) bound directly to whole cells of L. monocytogenes . This binding was blocked by apotransferrin indicating that the receptor can bind transferrin in either the iron-bound or iron-free form. Transferrin-binding was not host specific because both bovine and equine transferrin inhibited the binding of HRP-conjugated human transferrin. SDS-PAGE and Western blotting of bacterial surface extracts revealed the presence of a transferrin-binding protein of approximately 126 kDa.     

An iron-dependent mutant of Listeria monocytogenes of attenuated virulence

Abstract A bank of Tn 917 -insertional mutants from the facultative intracellular pathogen Listeria monocytogenes was screened by an original method based on bacterial growth on synthetic medium under iron-limiting conditions. One mutant, whose in vitro growth in synthetic medium was specifically dependent upon the availability of iron in its environment, was isolated and characterized. The insertional event occurred in a non-coding region, upstream of a rrn operon and located within a 1100-kb Not I fragment of the physical map, where the virulence genes already identified in L. monocytogenes were also present. Protein analysis by SDS-PAGE revealed a pleiotropic effect of the insertional event on cell-associated proteins, suggesting a polar effect of the transposon on adjacent unknown gene(s). The virulence in the mouse of this mutant was strongly impaired, although it was capable in vitro of growing intracellularly and of spreading from cell to cell, as shown by the production of lytic plaques on cell culture.     

Murine model of pregnancy-associated Listeria monocytogenes infection

Listeria monocytogenes has been recognized as a significant pathogen, occurring worldwide, capable of causing animal and human infections. In its most severe form, listeriosis is an invasive disease that affects immunocompromised patients. Additionally, pregnant women represent a high-risk group for L. monocytogenes infection. Abortion, stillbirth or severe neonatal infection can be the serious outcome of such an infection. In an experimental murine model of pregnancy-associated listeriosis we studied the impact of L. monocytogenes on the maternal immune response and pregnancy outcome. In comparison to virgin animals, pregnant mice mounted lower levels of protective cytokines and were unable to eliminate the pathogen. The impaired maternal immune response that has been found both on the systemic and local level, facilitated bacterial multiplication in the liver, placenta and ultimately in the fetal tissues. This resulted in severe necrotizing hemorrhagic hepatitis and Listeria-induced placental necrosis, increasing the incidence of postimplantation loss and poor pregnancy outcome.     

选择性增菌液对单核增生性李斯特氏菌检出效果的比较

为了了解食品中单核增生李斯特氏茵(Listeria monocytogenes)的污染状况,比较不同选择性增茵方法对单核增生李斯特氏茵的检出效果,并进一步比较不同增茵方法在不同类食品中检出单核增生李斯特氏茵的差异性,进而确定特定食品最合适的增茵方法,随机采集本市生鲜肉、水产品、果蔬及冷冻食品4类135份食品.采用国标LB二次增茵法、EB法、最新改良FDA法及Fraser肉汤增菌法进行增菌,采用PALCAM选择性平板进行分离,先用行标多重PCR法进行初步验证后再进行国标生化鉴定.4种方法共检出单核增生李斯特氏茵23株,其中LB二次增菌法检出5株、Fraser肉汤增菌法检出6株、EB法检出5株、最新改良FDA法检出7株.结论是4种方法总的检出率没有较大的差异性,但对于不同类食品的检出率有所不同.     

食品中单核增生李斯特氏菌检测研究进展

单核增生李斯特氏菌和李斯特菌病的危害近年来引起世界各国食品和卫生部门的广泛关注.关于如何找到一种快速、敏感、准确、合理的检验方法,是当今各国食品卫生部门亟待解决的重要研究课题.对该菌的传统分离方法、免疫学检测方法、核酸检测等方法的最新进展进行了综述,为进行该菌的准确、快速检测奠定了基础.     

Listeria monocytogenes as a probe of immune function.

For almost half a century, the mouse model of Listeria monocytogenes infection has been used to analyse both innate and adaptive components of immunity and to discover key immune genes. Vast accumulated knowledge about the disease in mice provides a unique framework for identifying and characterising immune molecules using a variety of experimental approaches. To illustrate the range of questions that can be addressed using modern genetics and genomics tools, the authors provide an overview of the analysis of components of immune signalling networks using the mouse model of L. monocytogenes infection.