棉铃虫作为单核细胞增生李斯特菌感染模型的初步研究

研究单核细胞增生李斯特菌(Listeria monocytogenes,简称Lm)对棉铃虫(Helicoverpa armigera)的致病性,探讨棉铃虫作为Lm感染模型的可行性。用野生株EGDe(中毒)、PrfA缺失株(EGDeΔprfA,弱毒)和PrfA组成型高表达株(EGDeΔprfA+pERL3-prfA~*,高毒)腹腔微量注射4龄棉铃虫幼虫,计算半数致死量(LD_(50))和虫体的载菌量,同时观测棉铃虫中肠细胞的病理变化以及血细胞包囊作用和数目。实验结果显示:①EGDe、EGDeΔprfA和EGDeΔprfA+pERL3-prfA~*的LD_(50)分别为2.56×10~3 cfu/mL、1.98×10~6 cfu/mL和6.63×10~2 cfu/mL。②感染Lm后,EGDe和EGDeΔprfA+pERL3-prfA~*在棉铃虫体内各部分的活菌数均有所上升,而EGDeΔprfA却显著下降。③中肠病理切片显示,Lm对棉铃虫幼虫中肠细胞的损伤程度与菌株的毒性高低正相关,并且随时间延长差异更为明显。④EGDeΔprfA+pERL3-prfA~*对棉铃虫血细胞的包囊作用的抑制作用最强,而EGDeΔprfA基本无抑制作用。⑤感染Lm后,棉铃虫的血细胞数量先急剧上升随后减少。72 h时,感染EGDe、EGDeΔprfA和EGDeΔprfA+pERL3-prfA~*的血细胞减少量分别为45%、27%和71%。结果表明,Lm不仅能成功侵染棉铃虫幼虫并致其死亡,且其半数致死量、其对棉铃虫中肠细胞的损伤程度以及对血细胞包囊作用和数量的影响等均与细菌毒力高低有较好的关联性,表明棉铃虫幼虫(至少是4龄棉铃虫幼虫)适合作为研究Lm致病机制的昆虫模型。     

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。     

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。     

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。     

单核细胞增生李斯特菌hfq基因缺失株的构建及其生物学特性

【目的】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)为革兰氏阳性的短杆菌,营腐生和寄生生活,是重要的食源性人兽共患病原菌,对低温、酸碱和高渗透压等环境具有较强的抵抗力。Lm在各种环境中适应、生存并表现致病力,是与调控因子的网络调控密切相关,本文初步研究了细菌调控因子hfq的生物学特性。【方法】利用同源重组技术对血清型为1/2 a的EGDe菌株进行hfq基因的敲除,对获得的突变株EGDe△hfq进行生化鉴定及生物学特性研究。【结果】实验结果表明:在4℃低温环境下,缺失株生长显著减慢(P<0.05);在含7%Na Cl高渗BHI培养基及含4.5%乙醇的BHI培养基中生长受到抑制;缺失株形成生物被膜的能力显著下降(P<0.05);对Caco-2细胞系的侵袭能力下降;与野生型菌株相比,EGDe△hfq对BALB/c小鼠的感染能力减弱、半数致死剂量提高。【结论】由此表明,Hfq蛋白对细菌抗胁迫、生物被膜形成及细菌毒力具有重要的调控作用。此缺失株的构建为进一步研究Hfq的功能提供了材料,为研究其在Lm胁迫环境生存及疾病预防控制奠定了基础。     

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。     

谷氨酸脱氢酶缺失对单核细胞增生李斯特菌生物被膜、毒力及胞外蛋白表达的影响

目的:探究谷氨酸脱氢酶缺失对单核细胞增生李斯特菌(Listeria monocytogenes,Lm)生物被膜、毒力及胞外蛋白表达的影响。方法:利用微孔板法,检测谷氨酸脱氢酶缺失对Lm生物被膜形成能力的影响;比较野生株、谷氨酸脱氢酶缺失株EGDeΔgdh A及回复菌株EGDeΔgdh A+p ERL3-gdh A的溶血活性和对棉铃虫幼虫的半数致死量,检测谷氨酸脱氢酶缺失对Lm毒力的影响;利用i TRAQ技术对Lm野生株EGDe与谷氨酸脱氢酶缺失株EGDeΔgdh A的胞外蛋白进行分离与鉴定,并对差异表达蛋白质进行生物信息学分析。结果:与野生株相比,缺失株形成的生物被膜量显著降低(P≤0.01),溶血活性下降,对棉铃虫幼虫的半数致死量上升了1.6倍;蛋白质组学结果显示,缺失谷氨酸脱氢酶后差异性表达的胞外蛋白有62个,其中47个蛋白质表达量下调,15个蛋白质表达量上调。进一步将这些差异表达蛋白质同COG数据库进行比对及功能聚类分析,结果显示,这些蛋白质的功能分别属于碳水化合物转运与代谢、核苷酸转运和代谢、能量合成与转运相关、转录及细胞膜细胞壁相关蛋白等16个功能类别;其中碳氮代谢及能量转运相关蛋白数量最多,为24个,占所鉴定出的差异蛋白质总量的38.71%。表明谷氨酸脱氢酶是单核细胞增生李斯特菌中碳氮代谢和能量转运中的关键酶。同时,发现有3个与细菌黏附及生物被膜形成相关的蛋白质表达量下降。以上结果较好地解释了EGDeΔgdh A在基础培养基中生长缓慢和生物量显著性下降,以及生物被膜形成能力降低的现象。结论:谷氨酸脱氢酶在单核细胞增生李斯特菌生物被膜、毒力及胞外蛋白表达方面有重要作用。     

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。     

基因dat对单核细胞增生李斯特氏菌的生物学特性影响

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是一种重要的食源性致病菌,其基因dat编码D-丙氨酸氨基转移酶,可将D-谷氨酸转变为D-丙氨酸,后者是细菌细胞壁肽聚糖的组成成分。在Lm野生株EGDe actA及inlB双基因缺失株(EGDeΔactAΔinlB)的基础上,利用同源重组的方法进一步构建了缺失基因dat的菌株(EGDeΔactAΔinlBΔdat),并对该缺失菌株生长状态、毒力基因表达水平、细胞侵袭及生物被膜的形成量等基本生物学特性进行研究,运用Real Time-PCR(RT-PCR)测定Lm毒力基因的相对表达量。结果表明,基因的缺失对突变菌株的生长能力以及生物被膜形成有重要的调控作用,并导致毒力基因srt A、plc A表达量下调,VIP、inlA表达量上调,对Coca-2细胞的侵袭无影响。此缺失株的构建为进一步研究基因dat的功能提供了材料。     

组氨酸氨解酶HutH对副溶血弧菌生物学特性及致病性的影响

组氨酸氨解酶(HutH)作为组氨酸代谢通路上的第一个代谢酶,控制细菌内部组氨酸的代谢。HutH在多数细菌中高度保守,参与细菌的能量代谢平衡。【目的】选取HutH作为研究对象,探究其对副溶血弧菌生物学特性以及致病性的影响。【方法】利用同源重组的方法构建缺失株ΔhutH和回补株CΔhutH。研究HutH对副溶血弧菌生长特性、组氨酸利用能力、组氨酸代谢相关基因表达水平、运动性、生物被膜、环境耐受、细胞毒性以及对小鼠毒力的影响。【结果】与野生型菌株相比,hutH基因缺失不影响副溶血弧菌的生长特性、耐酸耐碱能力、耐盐能力和群集运动。但ΔhutH在组氨酸作为唯一碳源的M9极限培养基中生长受到显著抑制。另外,我们证实,hutH基因缺失使组氨酸代谢操纵子内的相关基因转录水平显著下降,基因VP0889的表达水平提高。hutH基因缺失导致副溶血弧菌生物被膜形成能力下降、泳动能力下降、对HeLa细胞的毒性降低、对ICR小鼠的致死率显著降低。【结论】本研究表明,hutH基因缺失影响副溶血弧菌代谢组氨酸的能力,而且首次证实,HutH在副溶血弧菌的生物被膜形成、运动性和对小鼠毒力等方面具有重要作用,为从调控组氨酸...     

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.     

Suitability of the prfA gene, which encodes a regulator of virulence genes in Listeria monocytogenes, in the identification of pathogenic Listeria spp.

The pathogenesis of listerial infections is complex and involves a number of virulence factors expressed by virulent Listeria species. We have recently described a regulator gene, prfA, that positively regulates the expression of a number of virulence factors in Listeria monocytogenes. When the prfA gene was used as a DNA probe, we found it to be extremely specific for the pathogenic species L. monocytogenes. No reaction was obtained with strains of all other species of this genus. By using this information, an oligonucleotide primer pair was developed that specifically amplifies the prfA gene in L. monocytogenes strains of all known serotypes.     

进口水产品中单增李斯特菌的分子流行病学特点

摘要：[目的] 探明进口水产品中单增李斯特菌的污染状况、致病性和分子特征。[方法] 针对2007年7月至2008年11月间从29个国家进口的1275批水产品,进行单增李斯特菌鉴定、谱系与血清型分析、小鼠毒力试验与多位点序列分析。[结果] 检出单增李斯特菌33批次（2.6%）,其中以4b型为主（65.2%）,而1/2a型、1/2b型与1/2c型仅分别占13.0%、17.4%与4.4%。这些分离株对小鼠均具有与强毒参考株相当的毒力。基于actA-hisJ-ribC-sigB的多位点序列分析可将32个菌株分为23个序列型,分辨力达0.97。其中3个序列型包含3个以上分离株,其中序列型9属于流行性克隆I。[结论] 进口水产品中单增李斯特菌污染率与国内水产品相近,但血清型分布以4b型为主,且有流行性克隆I检出,因此要加强对进口水产品中单增李斯特菌的监测。     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow＇s Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow＇s milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes （iap, prfA, picA, hly, mpl, actA, plcB, InlA and lnlB） were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models （50% lethal dose： 10^8.14 VS. 10^5.49 and 10^6.73 VS. 10^1.9, respectively）. The genes prfA, picA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD （〉98%）. Genes iap, hly, plcB, lnlA and lnlB of L. monocytogenes 10403S had higher homology to those of strain EGD （〉98%） than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.     

Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow's Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow's milk,as screened in mouseand chicken embryonated egg models,was examined for virulence-related phenotypic traits.Correspondingvirulence genes (iap,prfA,plcA,hly,mpl,actA,plcB,InlA and InlB) were compared with L.monocytogenesreference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence.Al-though L.monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity,invitro growth and invasiveness and even had higher adhesiveness,faster intracellular growth and higherphospholipase activity in vitro,it was substantially less virulent than the strain 10403S in mouse and chickenembryo models (50% lethal dose:10~(8.14) vs.10~(5.49) and 10~(6.73) vs.10~(1.9),respectively).The genes prfA,plcA andmpl were homologous among L.monocytogenes strains H4,10403S and EGD (>98%).Genes iap,hly,plcB,InlA and InIB of L.monocytogenes 10403S had higher homology to those of strain EGD (>98%) than isolateH4.The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level,but 98.7% at the amino acid level.The actA gene of isolate H4 had deletions of 105 nucleotides correspondingto 35 amino acid deletions falling Within the proline-rich region.Taken together,this study presents someclues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletionmutations of actA.     

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(Listeria monocytogenes,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,比较野生株和缺失株对抗菌药物的耐药性;利用微孔板法观测突变株生物被膜形成能力的变化;利用平板泳动法研究菌株的泳动能力。【结果】与野生株相比,virAB缺失突变株对头孢类抗生素、Nisin和溴化乙锭的敏感性增加;当培养基中分别添加亚致死浓度的苯扎氯铵、卡那霉素和四环素时,突变株均表现出不同程度的生长缺陷。缺失virAB后菌株形成生物被膜的能力下降。【结论】VirAB在Lm对头孢类等抗菌药物的耐药及生物被膜形成方面具有重要作用。     

Listeria monocytogenes: comparative interpretation of mouse virulence assay

Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity. The availability of laboratory procedures to ascertain the pathogenic potential of L. monocytogenes bacteria would greatly enhance the control and prevention of listerial infections. As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L. monocytogenes virulence. The pathogenic potential of a given L. monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD(50)). In this report, we describe an alternative method [i.e., relative virulence (%)] that does not involve CFU estimation, and is comparable to LD(50) for interpretation of mouse virulence assay for L. monocytogenes. The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L. monocytogenes EGD) as reference. Besides providing a more direct interpretation in comparison with LD(50) values for mouse virulence assay, this method requires fewer dosage groups per L. monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories.