转铁蛋白受体在土拉弗朗西斯菌LVS入侵巨噬细胞期间的作用

评估转铁蛋白受体-1在土拉弗朗西斯菌LVS感染鼠巨噬细胞期间的作用.用表达GFP的土拉弗朗西斯菌LVS感染鼠巨噬细胞J774A.1.结合单抗的转铁蛋白受体-1分别用键合了Alexa594的羊抗鼠二抗显色,用小干扰RNA下调转铁蛋白受体-1的表达,进而用土拉弗朗西斯菌LVS分别感染转铁蛋白受体-1表达下调的细胞和转染无关siRNA的细胞,并进行细菌计数.分布在细胞膜上的转铁蛋白受体-1参与巨噬细胞对弗朗西斯菌的摄入.免疫印迹结果表明小于扰RNA对转铁蛋白受体-1的表达下调了大约75%.细菌入侵试验显示,在感染1 h时,转铁蛋白受体-1表达下调的细胞内细菌数量等同于对照(F=1.06.P=0.3265);而在感染24 h时,Tfr1下调样本中的细菌数量明显低于对照样本(F=24.12,P=0.0006).这些发现说明在感染早期转铁蛋白受体-1参与了细菌的摄入,这可能与弗朗西斯菌获取铁以利在胞内生存有关.转铁蛋白受体-1的下调不影响细菌的入侵,但抑制细菌在细胞内的增殖.     

细胞膜微结构域中胆固醇对小鼠巨噬细胞摄取土拉菌的作用

本文旨在探讨细胞膜微结构域中胆固醇在小鼠巨噬细胞摄取土拉弗朗西斯菌LVS株中的作用。用电穿孔法将穿梭质粒pFNLTP6 gro-gfp导入土拉弗朗西斯菌LVS株; 细胞胆固醇用菲律平Ⅲ染色, 结合单克隆抗体的小窝蛋白-1 用键合了Alexa 594 的羊抗鼠二抗显色; 用Z 轴电动聚焦的荧光显微镜分析土拉弗朗西斯菌LVS株感染; 用甲基-β-环糊精干扰富含脂质的胞膜, 损耗胆固醇, 以评估膜微结构域的损伤对土拉弗朗西斯菌入侵的影响, 菲律平Ⅲ用于检测胆固醇损耗的效果。结果显示, 细胞胆固醇在早期与摄取的土拉弗朗西斯菌疫苗株共定位, 膜微结构域相关成分小窝蛋白-1 与两者接触密; 土拉弗朗西斯菌入侵巨噬细胞需要胆固醇丰富的膜结构域。结果提示, 胆固醇丰富的膜微结构域和小窝蛋白-1 在土拉弗朗西斯菌早期进入巨噬细胞过程中发挥重要作用。     

肌动蛋白结合脂筏促进巨噬细胞摄入土拉弗朗西斯菌

观察土拉弗朗西斯菌LVS借助脂筏以肌动蛋白为动力被鼠巨噬细胞摄入的过程。细胞胆固醇用菲律平Ⅲ染色,结合神经节苷酯GM1的霍乱毒素B亚基用键合了Alexa 594的兔抗霍乱毒素B亚基二抗显色;肌动蛋白用键合了Alexa 594的鬼笔环肽显色。免疫荧光显微镜观察到脂筏成分中的胆固醇、神经节苷酯GM1均可与细菌共定位;胆固醇可与肌动蛋白共定位。随着感染时间的延长,细菌可离开脂筏。离开脂筏的细菌囊泡可与肌动蛋白共定位。这些发现提示肌动蛋白与脂筏结合,在弗朗西斯菌早期进入巨噬细胞期间发挥重要作用。     

鼠伤寒沙门菌感染巨噬细胞铁稳态相关基因mRNA表达谱

用荧光定量PCR法检测鼠RAW264.7巨噬细胞感染与未感染鼠伤寒沙门菌后18种铁穗态相关基因的表达,评估宿主与病原体相互作用中铁稳态效应。研究显示,活的鼠伤寒沙门菌感染巨噬细胞1 h后可以诱导转铁蛋白受体表达,引起细胞内动态铁池相关基因的mRNA水平上长。基因表达分析显示,沙门菌通过诱导铁氧还原酶(Steap3)、铁膜转运蛋白(Dmt1)、铁调节因子Tfr2/Hfe以及铁调节蛋白(Irp1和Irp2)的表达主动吸收铁,而经铁转运蛋白(Fpn1)的铁外流并无明显改变。沙门菌在感染后1h积极地驱动了转铁蛋白介导的铁吸收程序。     

TassC结合过氧化物酶体对早期受染巨噬细胞内的鼠伤寒沙门菌的影响

探讨鼠伤寒沙门菌在感染鼠巨噬细胞早期与细胞器的相互作用。用pTassC-GFP质粒转染鼠巨噬细胞RAW264.7,结合多抗的溶酶体标志物溶酶体相关膜蛋白-1用键合了Alexa594的羊抗鼠二抗显色,以观察标记了绿色荧光蛋白的TassC与溶酶体的关系;用pTassC-GFP和pDsRed2-Perxi质粒共转染RAW264.7细胞,以观察TassC-GFP与过氧化物酶体的关系;用SYTO42标记鼠伤寒沙门菌,感染用pTassC-GFP和pDsRed2-Perxi质粒共转染的RAW264.7细胞,以观察细菌与TassC和过氧化物酶体的关系。免疫荧光显示TassC-GFP不与鼠巨噬细胞RAW264.7中的溶酶体结合,但与标记了红色荧光的过氧化物酶体共定位;感染1 h的RAW264.7胞内SYTO42标记的鼠伤寒沙门菌吞噬泡可招募TassC-GFP和过氧化物酶体。这些发现提示在鼠伤寒沙门菌感染早期过氧化物酶体携带杀菌成分通过TassC介导可参与发挥一定的杀菌作用。     

小窝蛋白-1在细胞衰老及衰老相关疾病中的作用

胞膜小窝(caveolae)是细胞质膜内陷所形成的囊状结构.小窝蛋白(caveolin)是胞膜小窝区别于其它脂筏结构的特征性蛋白分子,维持胞膜小窝的结构和功能,包括3个家族成员小窝蛋白-1、小窝蛋白-2和小窝蛋白-3.其中,小窝蛋白-1是参与胆固醇平衡、分子运输和跨膜信号发放事件的主要结构成分,从而调节细胞的生长、发育和增殖．小窝蛋白-1在细胞衰老中起着重要调控作用,主要通过p53-p21及p16-Rb信号通路抑制细胞增殖、酪氨酸激酶的级联反应,调控粘连信号级联、胰岛素信号及雌激素信号系统等途径调控衰老进程．衰老过程中不同器官小窝蛋白-1变化趋势不尽一致．近年研究还发现,小窝蛋白-1与神经系统退行性疾病、糖尿病、动脉粥样硬化等衰老相关疾病密切相关,通过调节多条信号通路参与这些疾病的发生发展．本文结合最新研究进展,对小窝蛋白-1在细胞衰老进程的作用及参与衰老相关疾病进行综述．     

弗郎西斯菌分类鉴定与流行趋势的研究进展

弗朗西斯菌是一类革兰氏阴性染色的苛养性细菌。该菌属的模式菌种——土拉热弗朗西斯菌是人畜共患病土拉热病的病原体。弗朗西斯菌广泛存在于土壤、草丛、沼泽等自然环境和被感染的动物宿主体内,可以通过空气气溶胶、节肢动物叮咬以及直接接触受污染的环境、物品和饮食等多种方式传播。近年来,世界各地有大量新型的弗朗西斯菌种的发现报道。我们课题组于2008年始,从城市集中空调冷凝水系统中分离了多株弗朗西斯菌,并初步发现了两个弗朗西斯菌属的新种,其中广州弗朗西斯菌已获得正式命名。然而这些新细菌的鉴定方法,流行病学特征均不十分明确。本文简要综述了弗朗西斯菌的分类现状、分离鉴定方法等最新的研究进展情况,供国内学者参考。     

小窝蛋白-1在免疫调节中的作用

小窝（caveolae）是一类特殊的膜脂筏,富含鞘磷脂和胆固醇。小窝蛋白-1（caveolin-1）是小窝的标志蛋白质,分子量约22 kD。后者不但直接参与小窝结构的形成、膜泡运输、胆固醇稳态维持,还通过其脚手架结构域（caveolin scaffolding domain,CSD）与众多信号分子相互作用调控细胞的生长、发育和分化,最终影响机体的生理和病理过程。近年发现,小窝蛋白-1和胞膜窖不但存在于内皮细胞、脂肪细胞、血管平滑肌细胞和纤维细胞中,还广泛表达于免疫细胞中,参与调节免疫细胞活化引起的炎症应答反应。本文结合最新的研究进展和前期结果,简要综述小窝蛋白-1在巨噬细胞、T细胞、B细胞以及中性粒细胞等免疫细胞内的调节作用,以及在细菌感染如绿脓杆菌、沙门氏菌和克雷伯杆菌的炎症中的信号转导研究进展。     

土拉弗朗西斯菌胶体金免疫层析快速检测方法的建立

目的建立胶体金免疫层析技术快速定量检测土拉弗朗西斯菌。方法利用胶体金标记和双抗体夹心免疫层析技术,建立土拉弗朗西斯菌的快速检测方法,评价其特异性和敏感性,并拟合检测曲线进行定量检测。在面粉、饼干、果冻、梨汁等食品样品中添加土拉弗朗西斯菌的FopA蛋白模拟污染样品,评价该方法对固体、半固体、液体等食品样品的检测能力。结果该法可在10min内完成定性和定量检测,灵敏度为750ng／ml,线性范围750～24000ng/ml、回收率为56．7％-89．2％。结论所建立的检测土拉弗朗西斯菌的胶体金免疫层析方法,能快速、灵敏、特异、准确地检测样品中的土拉弗朗西斯菌,适用于现场快速检测。     

PHLCX Nflag3/小窝蛋白-1重组质粒的构建及融合基因在293T细胞中的表达

构建重组真核表达质粒PHLCX Nflag3／小窝蛋白-1,并在293T细胞中表达．用PCR的方法扩增cDNA文库中的人小窝蛋白-1基因,连接在真核表达栽体PHLCX Nflag3的短肽标签flag的下游,用限制性酶切和泖l序的方法鉴定;将重组质粒以脂质体法转染293T细胞,Western blotting法检测蛋白质的表达．结果显示,双酶切出现两个片段,分别与空栽体和人小窝蛋白-1的cDNA分子质量大小相符,测序结果符合人小窝蛋白-1的cDNA序列;Western blotting显示构建的新栽体能够在293T细胞中表达小窝蛋白-1／flag融合蛋白,表明已成功构建了能在293T细胞中高效表达小窝蛋白-1／flag融合蛋白的真核表达栽体PLHCX Nflag3／小窝蛋白-1．     

Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain

We used the killing of Galleria mellonella (Lepidoptera: Pyralidae; the greater wax moth) caterpillar by the live vaccine strain (LVS) of Francisella tularensis to develop an invertebrate host system that can be used to study F. tularensis infection and the in vivo effects of antibacterial compounds on F. tularensis LVS. After injection into the insect hemocoel, F. tularensis LVS, killed caterpillars despite the association of LVS with hemocytes. The rate of killing depended on the number of bacteria injected. Antibiotic therapy with ciprofloxacin, levofloxacin or streptomycin administered before or after inoculation prolonged survival and decreased the tissue burden of F. tularensis in the hemocoel. Delayed drug treatment reduced the efficacy of antibacterials and especially streptomycin. The G. mellonella-F. tularensis LVS system may facilitate the in vivo study of F. tularensis, efficacy with antibacterial agents.     

The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages

Intracellular killing of Francisella tularensis by macrophages depends on interferon-gamma (IFN-gamma)-induced activation of the cells. The importance of inducible nitric oxide synthase (iNOS) or NADPH phagocyte oxidase (phox) for the cidal activity was studied. Murine IFN-gamma-activated peritoneal exudate cells (PEC) produced nitric oxide (NO), measured as nitrite plus nitrate, and superoxide. When PEC were infected with the live vaccine strain, LVS, of F. tularensis, the number of viable bacteria was at least 1000-fold lower in the presence than in the absence of IFN-gamma after 48 h of incubation. PEC from iNOS-gene-deficient (iNOS-/-) mice killed F. tularensis LVS less effectively than did PEC from wild-type mice. PEC from phox gene-deficient (p47phox-/-) mice were capable of killing the bacteria, but killing was less efficient, although still significant, in the presence of NG-monomethyl-L-arginine (NMMLA), an inhibitor of iNOS. A decomposition catalyst of ONOO-, FeTPPS, completely reversed the IFN-gamma-induced killing of F. tularensis LVS. Under host cell-free conditions, F. tularensis LVS was exposed to S-nitroso-acetyl-penicillamine (SNAP), which generates NO, or 3-morpholinosydnonimine hydrochloride (SIN-1), which generates NO and superoxide, leading to formation of ONOO-. During 6 h of incubation, SNAP caused no killing of F. tularensis LVS, whereas effective killing occurred in the presence of equimolar concentrations of SIN-1. The results suggest that mechanisms dependent on iNOS and to a minor degree, phox, contribute to the IFN-gamma-induced macrophage killing of F. tularensis LVS. ONOO- is likely to be a major mediator of the killing.     

Uptake of serum-opsonized Francisella tularensis by macrophages can be mediated by class A scavenger receptors

The bacterium Francisella tularensis is highly infective, and this is one of the chief attributes that has led to its development as a bioweapon. Establishment of infection requires efficient uptake of F. tularensis by host macrophages, which provide a safe in vivo environment for F. tularensis replication. Little is known, however, about the cellular entry mechanisms employed by this organism. This report shows that efficient uptake of F. tularensis live vaccine strain (LVS) by macrophages is dependent on a heat-sensitive serum component and is mediated in part by types I and II class A scavenger receptors (SRA), demonstrating for the first time that SRA can act as a receptor for opsonized pathogens. Specifically, uptake of serum-opsonized LVS was partially blocked by general scavenger receptor inhibitors [fucoidan and poly(I)] and was largely inhibited by a specific function-blocking antibody against SRA. A role for SRA in LVS binding was confirmed by showing that ectopic expression of SRA in human embryonic kidney cells conferred the capacity for robust serum-dependent LVS binding. Finally, SRA-/- macrophages ingested significantly fewer LVS than did macrophages from wild-type mice. These findings support a novel role for SRA in innate immunity and suggest a potential therapeutic approach for modulating F. tularensis infection, namely, blocking SRA as a means of hindering F. tularensis access to its intracellular niche.     

Inhibition of Francisella tularensis LVS infection of macrophages results in a reduced inflammatory response: evaluation of a therapeutic strategy for intracellular bacteria

Francisella tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. A flow cytometric assay was developed to measure bacterial uptake, using a fluorescein isothiocyanate-labelled anti-F. tularensis lipopolysaccharide antibody in conjunction with antibodies to cell surface markers, in order to determine the specific cell phenotypes that were positive for the bacteria. Several phagocytic inhibitors were evaluated in macrophage cell lines and a lung homogenate assay to determine whether the uptake of F. tularensis strain LVS could be altered. Our data show that cytochalasin B, LY294002, wortmannin, nocodazole, MG132 and XVA143 inhibitors reduced LVS uptake by >50% in these assays without having significant cytotoxic effects. Furthermore, a reduction in the inflammatory cytokines monocyte chemoattractant protein-1, interleukin-6 and tumour necrosis factor-α was found in the supernatant of lung tissue infected with LVS when the inhibitory compounds were present. Similarly, there was an alteration in bacterial uptake and a reduction in the inflammatory cytokine response following the administration of wortmannin to LVS-infected mice. Although wortmannin treatment alone did not correlate with the enhanced survival of LVS-infected mice, these inhibitors may have utility in combination therapeutic approaches or against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.     

Francisella tularensis selectively induces proinflammatory changes in endothelial cells

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.     

Larval exposure to Francisella tularensis LVS affects fitness of the mosquito Culex quinquefasciatus

Francisella tularensis is an environmental bacterium capable of infecting a wide spectrum of species from mammals and birds to reptiles. It has been demonstrated that F. tularensis can invade and survive within protozoa, but an association with aquatic insects has not been thoroughly investigated. We examined the interaction of F. tularensis LVS biofilms and Culex quinquefasciatus larvae to determine the effects on larvae and adults. Our results demonstrate that F. tularensis LVS can form and persist as biofilms in natural water and that the mosquito larvae of C. quinquefasciatus readily feed on biofilm and planktonic forms of F. tularensis LVS. Larvae raised in both bacteria-only cultures suffered significant delays in pupation. Adults resulting from larvae continuously exposed to the bacteria had significantly reduced wing lengths in males and fecundity of both sexes. The bacteria may be exerting these effects through localization and persistence within the midgut and Malpighian tubule cells of the larvae. The study of oral acquisition of pathogens by insect larvae can significantly contribute to the study of environmental persistence of pathogens. We show that oral uptake of F. tularensis LVS by C. quinquefasciatus larvae results in not only larval effects but also has effects on adult mosquitoes. These effects are important in understanding both the ecology of tularemia as well as bacterial interactions with aquatic invertebrates.     

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.     

Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis

Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection.     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.