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。     

CodY在单核细胞增生李斯特菌运动和毒力方面的作用

目的:探究全局性转录调控因子CodY在单核细胞增生李斯特菌(Listeria monocytogenes,Lm)鞭毛运动和细菌毒力方面的作用。方法:通过同源重组的方法敲除Lm染色体上CodY的编码基因codY并成功构建缺失菌株的回复菌株;利用平板泳动法观测鞭毛运动的变化,RT-qPCR检测与鞭毛运动相关基因的转录表达;比较野生型菌株EGDe与CodY缺失菌株对细菌溶血活性、棉铃虫幼虫的半致死剂量和主要的毒力因子LLO和毒力基因调控蛋白PrfA转录表达的影响。结果:同野生型菌株相比,CodY缺失菌株鞭毛运动和相关基因,以及主要的毒力因子LLO和PrfA的转录表达显著降低(P≤0.01),溶血活性显著降低(P≤0.01),对棉铃虫幼虫的半致死剂量上升了5.8倍。结论:CodY在Lm鞭毛运动和细菌毒力调控方面具有重要作用。     

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.     

γ-辐射与常规方法灭活产单核细胞李斯特菌对小鼠免疫原性的比较

【目的】以产单核细胞李斯特菌(Listeria monocytogenes, LM)为模式菌, 比较γ-辐射及常规的热灭活、甲醛灭活制备的灭活李斯特菌对小鼠的免疫原性。【方法】以γ-辐射、热灭活和甲醛灭活法分别制备灭活的LM;腹腔注射BALB/c小鼠,ELISA检测各组灭活菌产生的抗体水平;观察野生型LM攻击后各组的保护效果,动态监测体内带菌情况;流式细胞仪分选免疫小鼠T细胞,以T细胞转移实验评价辐射灭活的LM产生的免疫应答。【结果】 辐射灭活组、热灭活组和甲醛灭活组产生的抗体水平分别为：1:1280, 1:640, 1:160;野生型细菌攻击后这3种灭活菌产生的保护率分别为：100%、35%、30%,其中免疫辐射灭活菌的小鼠能够较快清除攻击的细菌;辐射灭活李斯特菌能够激发产生T细胞保护性免疫应答。【结论】较之传统的灭活方法,利用γ-辐射制备的灭活LM免疫小鼠后能够产生较好的保护效果。     

ISA 61 VG佐剂增强单核细胞增生李斯特氏菌灭活疫苗的保护性免疫应答

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是重要的人兽共患李斯特氏菌病的致病菌,疫苗免疫是预防该病原菌感染的有效手段之一。本研究研制了添加矿物油佐剂MontanideTM ISA61VG的新型灭活细菌疫苗,并对其安全性和免疫应答特性进行了研究。结果表明,ISA 61 VG佐剂疫苗具有较好的安全性;诱导小鼠产生的抗李斯特氏菌溶血素O抗体滴度以及IgG2a/IgG1比值显著高于无佐剂免疫组;在致死剂量Lm攻毒下,能对小鼠提供100%的免疫保护。因此,ISA 61VG佐剂能显著增强灭活疫苗诱导宿主产生体液免疫和细胞免疫应答的能力,从而提高灭活疫苗的保护性免疫应答作用,是预防人和动物Lm感染的潜在疫苗候选株。     

利用转座子Tn917构建单核细胞增生李斯特菌菌膜形成突变株

单核细胞增生李斯特菌菌膜形成相关基因和调控因子的分离和鉴定是阐明其菌膜形成分子机理的基础。利用原生质体转化这一方式,将带有转座子Tn917的质粒pTV1OK成功地转进了单核细胞增生李斯特菌。通过诱导Tn917转座,得到单核细胞增生李斯特菌Tn917插入突变库,转座率为10-7。经96孔细胞培养板筛选发现,菌株LM49形成菌膜能力明显大于野生型。该菌株在细胞培养板中培养４d后形成的紫色圆环的颜色明显深于野生型。用Tn917特异引物进行PCR扩增,结果显示只有以该突变株的DNA为模板才能得到相应大小的扩增产物,证实该菌株基因组中有Tn917插入。Tn917的插入使菌株LM49的菌膜形成能力增强。     

单核细胞增生性李斯特菌prfA基因缺失株的构建及其生物学特性

【目的】单核细胞增生性李斯特菌(Lm)是人兽共患李斯特菌病的病原菌,其致病性与调控因子PrfA蛋白作用下毒力基因的表达有着密切关系,本文初步探讨了PrfA蛋白对细菌毒力因子的调控作用。【方法】利用同源重组技术对血清型分别为1/2a和4b的LM4、F4636进行prfA基因的敲除,并构建其回复突变株,对获得的突变株LM4ΔprfA、F4636ΔprfA进行生物学特性研究。【结果】实验结果表明:两株缺失株的溶血活性丧失、回复突变株的溶血活性得到恢复,突变株还丧失磷脂酶活性,黏附和侵袭特性显著下降(P<0.05),对BALB/c小鼠的半数致死剂量提高了105个数量级。【结论】由此表明,PrfA蛋白对hly、plcB、inl家族基因的表达及细菌毒力具有重要的调控作用。prfA基因缺失株的构建为进一步研究PrfA蛋白的调控功能提供了材料,为研究其在Lm致病性中的作用奠定了基础。     

VirAB在单核细胞增生李斯特菌耐药性及生物被膜形成中的作用

[背景]单核细胞增生李斯特菌(Listeriamonocytogenes,Lm)对一些临床常用抗生素、乳酸链球菌素(Nisin)等抗菌药物的敏感性下降,然而其背后的机制仍未完全阐明.[目的]调查转运蛋白VirAB在Lm对抗菌药物的耐药性及生物被膜形成中的作用.[方法]利用同源重组技术构建Lm基因缺失突变株,比较野生株和...     

prfA基因缺失影响单增李斯特菌诱导细胞凋亡的能力

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)入侵宿主细胞并带来危害,包含3个关键性环节,即宿主细胞的黏附和侵袭、细胞内增殖和运动、细胞间传播。每个环节的实现均需要不同毒力因子的调控,不同血清型Lm毒力的强弱取决于其毒力因子的活性,prfA基因作为Lm毒力的调控基因起到至关重要的作用。本研究使用单增李斯特菌野生株EGDe及其prfA基因缺失株EGDe-ΔprfA,以人结肠癌腺细胞Caco-2和人肝癌上皮细胞HepG2为研究对象,通过细胞侵袭实验和诱导凋亡能力探究prfA基因缺失对EGDe毒力的影响。溶血实验结果表明prfA基因的缺失并不会明显改变EGDe的溶血能力;侵袭实验表明突变株与EGDe野生株相比对Caco-2细胞和HepG2细胞侵袭能力分别下降了40%和30%;EGDe-ΔprfA处理组与EGDe处理组相比较,Caco-2细胞和HepG2细胞平均凋亡比例分别下降了35.8%和43.7%,说明prfA基因敲除后会降低细胞凋亡。本研究初步证实了prfA基因对EGDe毒力的调控作用,prfA基因缺失降低EGDe侵袭过程中的细胞凋亡,这对于了解EGDe的致病机理及进行相关研究有重要作用。     

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。     

A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.

The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.     

Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells

In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.     

Restricted translocation across the cell wall regulates secretion of the broad-range phospholipase C of Listeria monocytogenes

The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. Among the bacterial factors involved in escape from these vacuoles is a secreted broad-range phospholipase C (PC-PLC), the activation of which requires processing of an N-terminal prodomain. Mpl, a secreted metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. We previously showed that, during intracellular growth, bacteria maintain a pool of PC-PLC that is not accessible to antibodies and that is rapidly released in its active form in response to a decrease in pH. pH-regulated release of active PC-PLC is Mpl dependent. To further characterize the mechanism regulating secretion of PC-PLC, the bacterial localization of PC-PLC and Mpl was investigated. Both proteins were detected in the bacterial supernatant and lysate with no apparent changes in molecular weight. Extraction of bacteria-associated PC-PLC and Mpl required cell wall hydrolysis, but there was no indication that either protein was covalently bound to the bacterial cell wall. Results from pulse-chase experiments performed with infected macrophages indicated that the rate of synthesis of PC-PLC exceeded the rate of translocation across the bacterial cell wall and confirmed that the pool of PC-PLC associated with bacteria was efficiently activated and secreted upon acidification of the host cell cytosol. These data suggest that bacterially associated PC-PLC and Mpl localize at the cell wall-membrane interface and that translocation of PC-PLC across the bacterial cell wall is rate limiting, resulting in the formation of a bacterially associated pool of PC-PLC that would readily be accessible for activation and release into nascent secondary vacuoles.     

The metalloprotease of Listeria monocytogenes controls cell wall translocation of the broad-range phospholipase C

Listeria monocytogenes is a gram-positive bacterial pathogen that multiplies in the cytosol of host cells and spreads directly from cell to cell. During cell-to-cell spread, bacteria become temporarily confined to secondary vacuoles. The broad-range phospholipase C (PC-PLC) of L. monocytogenes contributes to bacterial escape from secondary vacuoles. PC-PLC requires cleavage of an N-terminal propeptide for activation, and Mpl, a metalloprotease of Listeria, is involved in the proteolytic activation of PC-PLC. Previously, we showed that cell wall translocation of PC-PLC is inefficient, resulting in accumulation of PC-PLC at the membrane-cell wall interface. In infected cells, rapid cell wall translocation of PC-PLC is triggered by a decrease in pH and correlates with cleavage of the propeptide in an Mpl-dependent manner. To address the role of the propeptide and of Mpl in cell wall translocation of PC-PLC, we generated a cleavage site mutant and a propeptide deletion mutant. The intracellular behavior of these mutants was assessed in pulse-chase experiments. We observed efficient translocation of the proform of the PC-PLC cleavage site mutant in a manner that was pH sensitive and Mpl dependent. However, the propeptide deletion mutant was efficiently translocated into host cells independent of Mpl and pH. Overall, these results suggest that Mpl regulates PC-PLC translocation across the bacterial cell wall in a manner that is dependent on the presence of the propeptide but independent of propeptide cleavage. In addition, similarly to Mpl-mediated cleavage of PC-PLC propeptide, Mpl-mediated translocation of PC-PLC across the bacterial cell wall is pH sensitive.     

Listeria monocytogenes evades killing by autophagy during colonization of host cells

Listeria monocytogenes is an intracellular pathogen that is able to colonize the cytosol of macrophages. Here we examined the interaction of this pathogen with autophagy, a host cytosolic degradative pathway that constitutes an important component of innate immunity towards microbial invaders. L. monocytogenes infection induced activation of the autophagy system in macrophages. At 1 h post infection (p.i.), a population of intracellular bacteria ( approximately 37%) colocalized with the autophagy marker LC3. These bacteria were within vacuoles and were targeted by autophagy in an LLO-dependent manner. At later stages in infection (by 4 h p.i.), the majority of L. monocytogenes escaped into the cytosol and rapidly replicated. At these times, less than 10% of intracellular bacteria colocalized with LC3. We found that ActA expression was sufficient to prevent autophagy of bacteria in the cytosol of macrophages. Surprisingly, ActA expression was not strictly necessary, indicating that other virulence factors were involved. Accordingly, we also found a role for the bacterial phospholipases, PI-PLC and PC-PLC, in autophagy evasion, as bacteria lacking phospholipase expression were targeted by autophagy at later times in infection. Together, our results demonstrate that L. monocytogenes utilizes multiple mechanisms to avoid destruction by the autophagy system during colonization of macrophages.     

Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes

Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways. We investigated whether L. monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells. In vitro culture of murine splenocytes with L. monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL). Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s). Examination of L. monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC). Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression. FasL-expressing cells induced by L. monocytogenes were capable of killing Fas-expressing target cells. Furthermore, L. monocytogenes infection results in upregulation of FasL on T cells in mice. These results describe a novel function for LLO and PC-PLC and suggest that L. monocytogenes may use these virulence factors to modulate the host immune response.     

FbpA, a novel multifunctional Listeria monocytogenes virulence factor

Listeria monocytogenes is a Gram-positive intracellular bacterium responsible for severe opportunistic infections in humans and animals. Signature-tagged mutagenesis (STM) was used to identify a gene named fbpA, required for efficient liver colonization of mice inoculated intravenously. FbpA was also shown to be required for intestinal and liver colonization after oral infection of transgenic mice expressing human E-cadherin. fbpA encodes a 570-amino-acid polypeptide that has strong homologies to atypical fibronectin-binding proteins. FbpA binds to immobilized human fibronectin in a dose-dependent and saturable manner and increases adherence of wild-type L. monocytogenes to HEp-2 cells in the presence of exogenous fibronectin. Despite the lack of conventional secretion/anchoring signals, FbpA is detected using an antibody generated against the recombinant FbpA protein on the bacterial surface by immunofluorescence, and in the membrane compartment by Western blot analysis of cell extracts. Strikingly, FbpA expression affects the protein levels of two virulence factors, listeriolysin O (LLO) and InlB, but not that of InlA or ActA. FbpA co-immunoprecipitates with LLO and InlB, but not with InlA or ActA. Thus, FbpA, in addition to being a fibronectin-binding protein, behaves as a chaperone or an escort protein for two important virulence factors and appears as a novel multifunctional virulence factor of L. monocytogenes.     

LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes

The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogen es EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes .     

Formation of D-alanyl-lipoteichoic acid is required for adhesion and virulence of Listeria monocytogenes

The dlt operon of Gram-positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.