Characteristics of induction of virulence factor expression by activated charcoal in Listeria monocytogenes

Activated charcoal has been previously shown to induce in vitro expression of virulence factors by Listeria monocytogenes. In trying to elucidate the nature of the charcoal action, we found that the treatment of brain heart infusion medium with activated charcoal followed by charcoal removal does not result in an increase of virulence factor expression. At the same time, the addition of fresh charcoal to the charcoal-treated medium induces expression, suggesting that the effect of activated charcoal cannot be explained only by changes in medium composition. In addition, we observed that activated charcoal induced expression of virulence factors even when L. monocytogenes was physically separated from charcoal particles by either a nitrocellulose membrane or a thin layer of agar. We propose that the interaction of charcoal with some listerial product(s) might be responsible for the effect observed.     

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.     

2016-2018年辽宁省食品中单核细胞增生李斯特菌污染情况及毒力基因的检测

目的了解辽宁省食品中单核细胞增生李斯特菌毒力基因携带特点,对该省食品中单核细胞增生李斯特菌的污染情况进行调查。方法依据GB 4789.30-2016《食品安全国家标准 食品微生物学检验 单核细胞增生李斯特氏菌检验》及采用PCR扩增技术检测的方法对2016-2018年采自该省15个监测点、收集的3 310份食品检出的47株单核细胞增生李斯特菌进行9种毒力基因检测。结果食品中单核细胞增生李斯特菌检出率为1.42%(47/3310),食品中单核细胞增生李斯特菌最少携带3种毒力基因,其中携带prfA、plcA、hly、mpl、plcB、inlA、inlB和iap八种毒力基因是该省食品中单核细胞增生李斯特菌的主要毒力基因型,达到检出菌总数的65.96%。结论研究结果证实辽宁省食品中存在单核细胞增生李斯特菌污染情况,应严格监控食品中单核细胞增生李斯特菌的携带情况。     

rmlB基因在单核细胞增生李斯特菌耐药性、生物被膜形成和毒力方面的作用

【目的】探究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)rmlB基因在细菌耐药、生物被膜形成和毒力方面的作用。【方法】通过同源重组的方法敲除Lm染色体上的rmlB基因,比较野生株与rmlB缺失株在耐药性方面的差异;利用微孔板法观测rmlB缺失菌株生物被膜形成能力的变化;利用RT-PCR检测缺失菌株中主要毒力基因转录表达,并观察rmlB缺失对细菌溶血活性的影响。【结果】同野生菌株相比,rmlB缺失菌株对头孢菌素和杆菌肽等作用位点在细菌细胞壁和细胞膜的敏感性显著增加(P≤0.01),生物被膜形成能力显著降低(P≤0.01),细菌主要毒力基因hly的转录表达及溶血活性也发生显著降低(P≤0.01)。【结论】rmlB基因在Lm生物被膜形成和耐受作用位点位于细胞壁和细胞膜的抗生素及细菌毒力方面具有重要作用。     

单增李斯特菌溶血素(LLO)的原核表达及其生物学活性鉴定

利用生物软件设计单增李斯特菌溶血素蛋白的基因hly的引物,通过PCR扩增hly基因,并将其克隆至PET28a(+)原核表达载体,转化大肠杆菌BL21进行优化表达。用镍柱纯化表达产物LLO,通过免疫印记鉴定其免疫原性,并通过溶血实验鉴定其溶血活性。琼脂糖凝胶电泳结果表明PCR扩增出1 590 bp的片段,经测序鉴定其序列同源性可达99%。SDS-PAGE结果表明诱导表达的产物大小约为58 kD,其最优化的表达条件是28°C下用0.1 mmol/L IPTG诱导6 h。Western blotting结果表明重组表达的LLO具有免疫原性;溶血实验表明重组表达的LLO具有较强的溶血活性,其溶血效价可达1:1 024。这为制备针对单增李斯特菌的单克隆抗体及其检测方法的建立奠定了基础。     

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。     

单核细胞增生李斯特菌中CcpA缺失株的构建及对毒力的影响

CcpA是革兰氏阳性菌中由ccpA基因编码的介导碳分解代谢物阻遏的全局调控因子。近年来的研究表明,CcpA不仅参与CCR效应,还直接或间接参与病原细菌毒力基因的表达调控。为探讨CcpA对单核细胞增生李斯特菌（Lm）毒力的影响,应用同源重组方法构建CcpA缺失菌株。以BLAB/c小鼠为实验动物模型,检测野生株EGDe和缺失株EGDeΔccpA侵染小鼠后的半数致死剂量 LD₅₀和肝脾细菌载菌量,观察小鼠肝脏和脾脏的病理形态变化。结果显示：缺失CcpA后,Lm的LD₅₀降低了10倍,虽然肝脾细菌载菌量没有显著变化,但EGDe△ccpA对小鼠肝和脾的损害更为严重,表明CcpA缺失增强了细菌的毒力,CcpA 对Lm 毒力基因的表达可能具有间接或者直接的调控作用。     

毒力基因调控蛋白PrfA促进单核细胞增生李斯特菌生物被膜的形成

单核细胞增生李斯特菌(Listeria monocytogenes,LM)是重要的革兰氏阳性食源性致病菌,易在食品以及各种食品加工、运输和保藏设备的接触面形成生物被膜,从而具有更强的抗逆性而难以彻底清除,因此成为食品卫生安全的重要隐患.PrfA是LM毒力基因转录表达的重要调控因子,通过比较研究LM野生株(EGD和EGDe)、PrfA缺失株(EGDAprfA和EGDeAprfA)、无害李斯特菌(Listeria innocua,LI),携带组成性表达PrfA蛋白的重组无害李斯特菌(LI-pERL3-prfA*)以及重组单核细胞增生李斯特菌(EGDeΔprfA-pERL3-prfA*)生物被膜形成能力的差异,探讨LM重要的毒力调控蛋白PrfA对生物被膜形成的影响.实验结果显示:LM野生株具有较强的生物被膜形成能力,而LI形成生物被膜的能力最弱;PrfA的缺失能降低LM生物被膜的形成能力;组成性高量表达PrfA蛋白可以回复EGDeΔprfA的生物被膜形成能力,但对LI没有增强作用.以上实验结果表明:PrfA在LM生物被膜形成中具有重要的促进作用.     

Isolation and characterization of a Listeria monocytogenes mutant strain hyperproducing virulence factors

A mutant strain characterized by hyperproduction of a number of cell wall and supernatant proteins was isolated after N-methyl-N′-nitro-N-nitrosoguanidine treatment of Listeria monocytogenes strain NCTC10527. Several of these proteins were identified as virulence factors. When a wild-type strain was grown in the presence of activated charcoal it was shown to exhibit the same protein pattern as the isolated mutant.     

Comparative assessment of acid, alkali and salt tolerance in Listeria monocytogenes virulent and avirulent strains

Listeria monocytogenes is an opportunistic bacterial pathogen of man and animals that has the capacity to survive under extreme environmental conditions. While our knowledge on L. monocytogenes and its ability to sustain within wide pH and temperature ranges and salt concentrations has been largely built on the virulent strains of this species, relatively little is known about avirulent strains in this regard. In this study, we extend our analysis on avirulent L. monocytogenes strains. By subjecting three virulent (EGD, 874 and ATCC 19196) and three avirulent (ATCC 19114, HCC23 and HCC25) strains to various pH and salt concentrations, it was found that L. monocytogenes recovered well after treatment with 100 mM Tris at pH 12.0, and to a lesser extent at pH 3.0. Interestingly, avirulent L. monocytogenes strains showed a somewhat higher tolerance to alkali than virulent strains. This unique feature of avirulent L. monocytogenes strains may potentially be exploited for the development of a rapid technique for differentiation between avirulent and virulent strains. Furthermore, all L. monocytogenes strains tested were resistant to saturated NaCl (about 7 M, or 40% w/v) for a long period of time (20 h and possibly longer). Together, these results highlight that acid, alkali, and/or salt treatments commonly used in food product processing may not be sufficient to eliminate L. monocytogenes, and therefore stringent quality control measures at the beginning and end of the food manufacturing process is essential to ensure that such food products are free of listerial contamination.     

Influence of immunomodulation on the development of Listeria monocytogenes infection in aged guinea pigs

We investigated the impact of immunomodulation on the development of listeriosis within an aged population of guinea pigs after an intragastric challenge with Listeria monocytogenes. Supplementation with vitamin E for 35 days significantly increased the level of cytotoxic T cells (CD8(+)), while treatment with cyclosporin A resulted in a 25% decrease of CD8(+) T cells. In the animals receiving the low dose (10(2) CFU) of L. monocytogenes, 50% of the control-group animals became infected. Only 22% of animals receiving the orthomolecular dose of vitamin E became infected, whereas animals that were immunosuppressed had an infection rate of 89%. In the immunosuppressed group three animals (16%) developed listerial infection with a quantifiable bacterial level of 0.3-3 log CFU g(-1) of organ in the spleen and liver. In the high-dose study, the population of L. monocytogenes was consistently 1 log CFU g(-1) lower in the spleen or liver of the vitamin E-supplemented group, compared with the control and cyclosporin A-treated animals. At day 4, a significant increase in the levels of CD8(+) during listerial infection occurred in vitamin E-supplemented animals, suggesting an increased ability to produce CD8(+) T cells. The results suggest that immunomodulation of the host can influence listerial infection within an aged population of guinea pigs.     

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.     

Impact of selected Lactobacillus and Bifidobacterium species on Listeria monocytogenes infection and the mucosal immune response

The pathogenesis of Listeria monocytogenes depends on its ability to attach to and invade the gastrointestinal epithelium and subsequently withstand the host immune response. Despite a thorough understanding of the intracellular phase of infection, relatively little is known about how the pathogen behaves in the gastrointestinal tract and whether it is affected by the presence of host commensal microbiota. Lactobacillus and Bifidobacterium are two important genera of the human gut microbiota proposed to possess probiotic effects. Here we demonstrate that probiotic bacteria significantly inhibit subsequent listerial infection in an in vitro C2Bbe1 epithelial cell model. In the case of Lactobacilli, inhibition was due to a combination of acid production and secretion of an as yet unidentified protein. In the case of Bifidobacterium, inhibition was attributable to an extracellular proteinaceous secreted compound. In addition, we observed a significant reduction in interleukin-8 and an increase in IL-10 cytokines secreted from epithelial cells following probiotic pretreatment and subsequent infection with Listeria. A reduction in the infection of epithelial cells and an altered mucosal immune response suggests that probiotic bacteria could be of therapeutic benefit against listerial infection. This study infers a role for probiotic bacteria as an antagonist of Li. monocytogenes infection.