毒力因子InlA和InlB缺失影响单增李斯特菌侵袭宿主细胞和诱导凋亡的能力

单核细胞增生性李斯特菌(Listeria monocytogenes,LM)是一种毒力较强、人畜共患的食源性致病菌,其具有穿透宿主屏障、胞内寄生的特点,因而致死率较高,被其污染的食品容易引发严重的食品安全问题。本研究使用前期构建的LM重要的毒力因子内化素A(internalin A,InlA)和InlB基因缺失菌株,以人结肠癌腺细胞Caco-2和人肝癌上皮细胞Hep G2为研究对象,探究InlA和InlB缺失对LM侵袭宿主细胞和诱导细胞凋亡的影响。实验结果表明,InlA和InlB的缺失使LM侵袭宿主细胞的能力明显降低(P0.05),与野生株相比侵袭量下降超过50%,同时也降低了其诱导宿主细胞凋亡的能力(P0.05),与野生株相比凋亡细胞的比例下降幅度达到30%~50%。本实验确定了InlA和InlB在LM侵袭宿主细胞和诱导细胞凋亡的过程中具有重要的作用,有助于对LM的致病机理以及引起宿主相关免疫反应、诱导细胞凋亡相关分子机制的深入研究。     

应用PCR技术快速测定食品中单核细胞增生李斯特菌毒力

目的：采用PCR 技术准确、快速测定单核细胞增生李斯特菌(单增李斯特氏菌)分离株的毒力基因,鉴别强毒株和弱毒株或无毒株,以有效地限制李斯特氏菌病的传播。方法：以单增李斯特氏菌相关毒力基因(hly、plcB、inlA、inlB、inlC、inlJ、prfA)设计引物,检测重庆市2007 - 2009 年分离的40 株单增李斯特氏菌分离株中的毒力基因携带率,并进行小鼠毒力实验。结果：40 株分离株中有15 株7 种毒力基因检测结果均为阳性,6 株hly 基因阴性,4 株plcB 基因阴性,4 株prfA 基因性,5 株inlA、inlB 基因阴性,11 株inlC 基因阴性,9 株inlJ基因阴性,1 株inlA、inlB、inlC、inlJ 基因均为阴性。4 株分离株的毒力与标准菌株ATCC191161E 相当,小鼠LD50 在1.2 × 108～6.0 × 108CFU/mL 之间,为强毒株。筛选出弱毒株09-132,LD50 为1.7 × 1011CFU/mL。结论：重庆市存在发生李斯特菌食物中毒的潜在危险,内化素基因(inlA、inlB、inlC、inlJ)的缺失可能是导致菌株毒力降低的原因,而prfA 和plcB 基因与菌株毒力的相关性较小。     

单增李斯特菌prfA基因缺失菌株的构建及其生物学特性鉴定

单核细胞增生性李斯特菌(Listeria monocytogenes,Lm)是一种风险较高的食源性致病菌,其绝大多数毒力基因的表达均受到由prfA基因编码的PrfA(positive regulatory factorA)蛋白全部或部分调控。使用同源重组的方法敲除Lm野生菌株EGDe的prfA基因,并通过测定生长曲线、毒力基因表达和侵袭Caco-2细胞能力等探讨单缺失菌株EGDe-ΔprfA生物学特性。通过测定生长曲线显示prfA敲除株和野生型EGDe二者生长状态无差异;运用实时定量荧光聚合酶链式反应检测EGDe-ΔprfA的主要毒力基因表达,结果显示plcA、plcB毒力基因的表达量分别上升至原来的2.5倍和2倍,inlA、inlB、inlC、actA、vip等均呈下降趋势,prfA基因表达量趋向于零;侵袭Caco-2细胞结果显示EGDe-ΔprfA侵袭数为野生株的1/5。该基因缺失菌株的构建及生物学特性研究对食源性致病菌EGDe致病机理研究具有重要意义并且提供了重要材料。     

双基因敲除减毒单增李斯特菌(ΔactA/ΔinlB)的构建及其生物学初步鉴定

减毒单增李斯特菌具有成为疫苗活载体的潜力,可同时引起MHCⅠ和MHCⅡ类抗原递呈系统,具有强烈激发CD8+和CD4+细胞免疫的能力。构建毒力基因缺失菌株进而评估其生物活性对于其作为疫苗活载体的开发尤为重要。本文采用同源重组技术构建单缺失菌株Lm-△act A和双缺失菌株Lm-△act A△/inl B,从生长状态、毒力基因表达和对Hep G2细胞侵袭能力方面探讨减毒菌株生物学特性。生长活性测定显示两种减毒菌株和野生型Lm(EGD-e)三者生长状况无差异;实时定量PCR结果显示act A基因缺失后,inl A基因表达水平上升两倍;act A和inl B基因双缺失后,plc B和hly基因的表达水平都有较大幅度的上升;侵袭Hep G2细胞结果显示act A基因缺失后侵袭能力增强、act A和inl B基因共同缺失后侵袭能力减弱。因此,减毒菌株的构建及其生物特性研究不仅对食源性致病菌Lm致病机理具有重要意义,也为构建预防人类和动物疫病的疫苗载体奠定了基础。     

单增李斯特菌nox基因的克隆、表达以及功能

以Gen Bank中报道的单增李斯特菌(Listeria monocytogenes,Lm)野生型菌株EGDe的nox基因(Gen Bank ID:986631)为研究对象,探讨在高等动植物中普遍存在的烟酰胺腺嘌呤二核苷酸磷酸氧化酶在Lm中是否也可以介导产生活性氧(reactive oxygen species,ROS)。首先通过诱导nox基因在BL21中表达产生Nox蛋白,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blot鉴定该蛋白质分子质量;然后构建过表达菌株EGDe-nox,测定ROS产生情况,并使用实时荧光定量-聚合酶链式反应检测nox基因的过表达对Lm毒力基因表达的影响。结果表明,经鉴定Nox蛋白分子质量约为33 k D,过表达菌株EGDe-nox与对照组EGDe的ROS产生量相比并无多大变化,nox基因的过表达会导致与侵袭相关的基因act A、inlA和inlB以及毒力基因prfA的表达上调。由此推测,该Nox蛋白不能独立主导ROS的产量,但其过表达却可以增强毒力基因表达上调。本研究为继续探讨细菌中Nox的作用提供一定的参考依据。     

单增李斯特菌(EGDe)sigB基因缺失株的构建及其生物特性的初步鉴定

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是一种人畜共患的食源性致病菌,在感染过程中可以穿越人体肠道屏障、胎盘屏障及血脑屏障,进而引发肠胃炎、脑膜炎及孕妇流产等疾病。Sigma B(sigB)基因编码产生的sigma B因子是许多革兰氏阳性菌对环境胁迫产生应答反应的主要调控因子,并且直接或间接的调控Lm中相关重要毒力基因如prf A、inl A等的表达。本文通过同源重组技术将Lm野生菌株EGDe的sigB基因敲除,并且对缺失菌株的生长曲线,inl A、inl B、prf A等13种Lm的毒力基因表达水平及对肠道上皮细胞Caco-2的侵袭进行了研究。实验表明,EGDe-ΔsigB基因缺失菌株生长速度与EGDe基本一致;sigB基因的缺失造成inl A和inl B基因表达水平的大幅度下调,但act A和plc A等毒力基因却上调4~5倍,说明sigB基因对于单核增生性李斯特菌的一些毒力基因表达影响比较大;Caco-2细胞的侵袭实验显示EGDe-ΔsigB基因的缺失对Caco-2细胞侵袭能力的下降。     

多重PCR-变性高效液相色谱快速检测单核细胞增生李斯特菌毒力基因方法的建立

建立单增李斯特菌的多个靶基因快速检测手段,提高检测的准确性。方法 根据单增李斯特菌4个毒力基因(hly、prfA、inlA、inlB)设计引物,通过优化引物浓度和引物组合,进行多重PCR扩增,产物经变性高效液相色谱(DHPLC)进行快速检测。结果 出峰顺序依次为inlB、hly、inlA、prfA,扩增片段大小为146、210、255、388bp,此方法具有良好的特异性,灵敏度可达到280cfu/ml。结论 本方法可以满足实际工作中食品微生物检测的要求。     

单核细胞性李斯特菌基因dal的敲除及其生物学特性初步分析

在单核细胞性李斯特菌(Listeria monocytogenes)野生株EGDe act A及inl B双基因缺失株(EGDeΔact AΔinl B)的基础上,利用同源重组的方法进一步构建了缺失营养基因dal的菌株(EGDe Δact AΔinl BΔdal),并对该缺失菌株生长状态、毒力基因表达水平、生物被膜的形成量及细胞侵袭等方面作进一步分析。结果显示,37℃摇床培养6 h后,缺失株的菌浓度显著低于EGDe Δact AΔinl B(P0.001),培养基中补充D-丙氨酸的缺失株生长速率与亲本株相比无显著差异;实时荧光定量聚合酶链式反应结果显示,缺失株的sig B基因表达水平变化最明显(P0.01),约下调90%;缺失株生物被膜形成量显著增加(P0.05),培养基补充D-丙氨酸后缺失株生物被膜的生成量与亲本株相比无差异;对Coca-2细胞的侵袭无影响,表明该基因对细菌生长能力及生物被膜形成具有重要的调控作用,并不影响菌株对细胞的侵袭力。此缺失株的构建为进一步研究基因dal的功能提供了理论支持。     

单核增生性李斯特菌hly缺失菌株的构建及生物特性的初步鉴定

为研究单核增生性李斯特菌(Listeria monocytogenes,Lm)hly基因对毒力的影响,利用同源重组原理,使用穿梭载体,构建单核细胞增生性李斯特菌野生菌株EGD-e的hly基因缺失菌株EGD-eΔhly。细菌活性实验证明缺失菌株生长状态与亲本无差异,但EGD-eΔhly丧失溶血活性,细胞侵袭能力降低约90%,在10~7 cells/m L腹腔注射条件下不表现动物毒性。在转录水平上EGD-eΔhly的毒力基因inl C、prf A的表达量分别下降了81%和76%,act A、plc B的表达量分别提高了2.7倍和1.8倍。hly缺失菌株的成功构建及生物特性的初步研究结果为研究Lm致病机理提供依据。     

鲜活贝类中单核细胞增生李斯特菌的分离鉴定及毒力基因与耐药性分析

目的研究采自产地的鲜活贝类中单核细胞增生李斯特菌(单增李斯特菌)的污染状况、毒力基因分布与耐药性。方法对采自产地的200份鲜活贝类,按照GB/T 4789.30—2010《食品安全国家标准食品卫生微生物学检验单核细胞增生李斯特氏菌检验》进行单增李斯特菌的分离与鉴定;采用Kirby-Bauer纸片扩散法测定分离株的耐药性;通过PCR法分析分离株9个毒力基因(包括prfA、plcB、hly、actA、iap、inlA、plcA、mpl、inlB)。结果在200份鲜活贝类中,共检出阳性样品4份(2.0%);有1株缺失inlB基因,1株缺失mpl基因;分离株对氨苄青霉素、庆大霉素等一线临床治疗药物敏感,但有2株对四环素和复方新诺明同时耐药,1株对复方新诺明和氧氟沙星耐药。结论采自产地的鲜活贝类存在单增李斯特菌的污染,分离株毒力基因有一定程度缺失,提示初级水产品中的单增李斯特菌污染需引起关注,并且需要继续加强食品中该菌的耐药性监测。     

Plant-derived antimicrobials reduce Listeria monocytogenes virulence factors in vitro, and down-regulate expression of virulence genes

Listeria monocytogenes (LM) is a major foodborne pathogen causing septicemia, meningitis and death in humans. LM infection is preceded by its attachment to and invasion of human intestinal epithelium followed by systemic spread. The major virulence factors in LM include motility, hemolysin and lecithinase production. Reducing LM attachment to and invasion of host tissue and production of virulence factors could potentially control listeriosis in humans. This study investigated the efficacy of sub-inhibitory concentrations (SICs, concentrations not inhibiting bacterial growth) of three, generally regarded as safe (GRAS)-status, plant-derived antimicrobial compounds in reducing LM attachment to and invasion of human colon adenocarcinoma (Caco-2) and human brain microvascular endothelial cells (HBMEC). Additionally, the effect of these compounds on the aforementioned LM virulence factors was studied. The compounds and their respective SICs used relative to their MICs were trans-cinnamaldehyde (TC 0.50mM, 0.75mM with the MIC of 0.90mM), carvacrol (CR 0.50mM, 0.65mM with the MIC of 0.75mM), and thymol (TY 0.33mM, 0.50mM with the MIC of 0.60mM). All three-plant antimicrobials reduced LM adhesion to and invasion of Caco-2 and HBMEC (p<0.05). The compounds also decreased LM motility, hemolysin production and lecithinase activity (p<0.05). Real-time PCR data revealed that TC, CR, and TY down-regulated the expression of LM virulence genes by >3.0 folds compared to controls (p<0.05). Results suggest that TC, CR, and TY could potentially be used to control LM infection; however, in vivo studies are necessary to validate these results.     

Induction of apoptosis by tomato using space mutation breeding in human colon cancer SW480 and HT‐29 cells

BACKGROUND: As far as we know, there have been no reports concerning the functional characteristics of tomatoes using space mutation breeding. The aim of this study was to evaluate the anti‐colon cancer effect of tomatoes M1 and M2 using space mutation breeding. RESULTS: In the present study, obvious anti‐cancer activity was shown with tomato juice of M1 and M2 and their parent CK treatment in colon cancer cell lines SW480 and HT‐29 in cell growth inhibition. In addition, SW480 cells were more sensitive to M1 and M2 than HT‐29 cells in cell apoptosis. Furthermore, M1 and M2 induced cell cycle arrest both in G0–G1 and G2/M phases. CONCLUSION: These data suggest that consumption of tomato using space mutation breeding may provide benefits to inhibit growth of colon cancer cells. Therefore, tomato production using space mutation breeding may be a good candidate for development as a dietary supplement in drug therapy for colon cancer. Copyright © 2010 Society of Chemical Industry     

Vitamin B6 suppresses serine protease inhibitor 3 expression in the colon of rats and in TNF-α-stimulated HT-29 cells

Scope: Previous reports in the areas of animal studies and, recently epidemiology, have linked anti‐tumorigenic and anti‐inflammatory effects to dietary vitamin B6. This study investigated the molecular mechanism of these effects of vitamin B6. Methods and results: DNA microarray analysis was used to obtain information on changes in colon gene expression from vitamin B6 (pyridoxine) repletion in vitamin B6‐deficient rats. Pyridoxine supplementation down‐regulated the inflammatory molecule, serine protease inhibitor clade A member 3 (SPI‐3) mRNA expression in the colon. This study also showed that tumor necrosis factor α (TNF‐α) induced SPI‐3 mRNA expression in HT‐29 human colon cancer cells, and vitamin B6 (pyridoxal hydrochloride) pretreatment of HT‐29 cells inhibited TNF ‐induced mRNA expression of SPI‐3. Vitamin B6 inhibited TNF‐α‐induced NF‐κB activation via suppression of IκBα degradation in HT‐29 cells. HT‐29 cells stably expressing epitope‐tagged ubiquitin were generated and vitamin B6 pretreatment was shown to inhibit ubiquitination of the IkB protein in response to TNF‐α‐i. Conclusion: Vitamin B6 suppressed SPI‐3 expression in the colon of rats and in TNF‐α‐stimulated HT‐29 cells. Further, this study showed a possible role of vitamin B6 in the regulation of protein ubiquitination.     

Incongruence between the cps type 2 genotype and host-related phenotypes of an Enterococcus faecalis food isolate

Enterococcus faecalis is a nosocomial opportunistic pathogen, but is also found in fermented food products where it plays a fundamental role in the fermentation process. Previously, we have described the non-starter E. faecalis cheese isolate QA29b as harboring virulence genes and proven to be virulent in Galleria mellonella virulence model. In this study, we further characterized this food strain concerning traits relevant for the host-pathogen relationship. QA29b was found to belong to sequence type (ST) 72, a common ST among food isolates, and thus we consider it as a good representative of food E. faecalis strains. It demonstrated high ability to form biofilms, to adhere to epithelial cells and was readily eliminated by J774.A1 macrophage cells. Despite carrying the cps locus associated with the capsular polysaccharide CPS 2 type, cps genes were not expressed, likely due to an IS6770 inserted in the cpsC-cpsK promoter region. This work constitutes the first study of traits important for interaction, colonization and infection in the host performed on a good representative of E. faecalis food isolates. Reported results stress the need for a reliable serotyping assay of E. faecalis, as cps genotyping may not be reliable. Overall, QA29b characterization shows that despite its virulence potential in an insect model, this food strain is readily eliminated by mammalian macrophages. Thus, fine tuned approaches combining cellular and mammalian models are needed to address and elucidate the multifactorial aspect of virulence potential associated with food isolates.     

Anti‐inflammatory and anticancer activities of water‐soluble peptide extracts of buffalo and cow milk Cheddar cheeses

This article aimed to assess the anti‐inflammatory and anticancer potential of water‐soluble peptide (WSP) extracts from buffalo and cow milk Cheddar cheeses. Anti‐inflammatory activity was evaluated on the basis of nitric oxide (NO) production in lipopolysaccharide‐stimulated macrophage (RAW‐264.7) cells. A cell viability assay, cell cycle arrest and apoptosis were performed to explore anticancer activity in a colon cancer model (HT‐29). The WSP extracts of both Cheddar cheeses effectively inhibited NO production in activated macrophages. Maximum growth inhibition was observed in the HT‐29 cells at concentrations of 400 and 500 μg/mL. A significant increase in cell population at G₀/G₁ phase of the cell cycle was observed. Moreover, the WSP extracts also induced extensive apoptosis in colon cancer cells.     

原儿茶醛对阪崎克罗诺肠杆菌毒力因子的抑制作用

探究植物源活性物质原儿茶醛对阪崎克罗诺肠杆菌泳动运动能力、生物被膜形成能力、黏附和侵入Caco-2细胞能力及9 种与菌体相关毒力基因转录的影响。结果表明,原儿茶醛对6 株阪崎克罗诺肠杆菌实验菌株的最小抑菌质量浓度（minimal inhibitory concentrations,MIC）和最小杀菌质量浓度均为2 mg/mL,对阪崎肠杆菌ATCC29544亚抑制浓度分别为1/200、1/100 MIC和1/50 MIC。亚抑制浓度的原儿茶醛能够抑制阪崎克罗诺肠杆菌在琼脂软平板中泳动运动能力;减弱菌体在12 ℃和25 ℃下不同时间段（24、48、72 h）生物被膜的形成能力;降低细菌黏附及侵入Caco-2细胞能力;下调9 种与菌体相关毒力基因的转录量。研究结果表明原儿茶醛能够抑制阪崎克罗诺肠杆菌多种毒力因子,其有潜力作为抗生素补充剂或抗毒性物质预防及控制阪崎克罗诺肠杆菌引起的相关感染。     

Cranberry extract and quercetin modulate the expression of cyclooxygenase‐2 (COX‐2) and IκBα in human colon cancer cells

BACKGROUND: Cranberry (Vaccinium marcocarpon) fruit and quercetin, a major flavonoid found in cranberries, are likely contributors to chemoprevention, and their anti‐inflammatory activities may play a potential role in colon cancer prevention. The aim of this study was to examine the effect of cranberry extract and quercetin on basal expression of cyclooxygenase‐2 (COX‐2) and IκBα as well as the effect on phorbol 12‐myristate 13‐acetate (PMA)‐induced COX‐2 expression in colon cancer cells. RESULTS: HT‐29 human colon adenocarcinoma cells were treated with various concentrations of cranberry extract or quercetin and/or PMA, and the protein expression of COX‐2 and IκBα was determined. The results indicated that cranberry extract and quercetin decreased COX‐2 expression and suppressed degradation of IκBα in unstimulated cells. In PMA‐stimulated cells, cranberry extract was also able to decrease COX‐2 expression and suppress degradation of IκBα. CONCLUSION: The results suggest that a possible mechanism involved in the anti‐cancer activity of cranberry and quercetin is partly mediated through its anti‐inflammatory action. These findings indicate that cranberry and quercetin may reduce the risk of colon cancer possibly by suppressing inflammatory responses. Copyright © 2008 Society of Chemical Industry