【作者】 达来宝力格；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2008， 博士

【摘要】 胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)是猪传染性胸膜肺炎(porcine contagious pleuropneumonia,PCP)的病原菌,引起猪的一种高度接触传染性呼吸道疾病,给世界养猪业造成严重的经济损失。APP是猪呼吸道的专性寄生菌,通过空气和直接接触来传播。由于APP血清型众多,各血清型之间交叉保护力较低,传统的灭活疫苗和亚单位疫苗的效果均不理想。因此,迫切需要更安全、高效的新型疫苗来预防和控制该传染病的发生与流行。毒力因子的全面鉴定是病原菌致病机理研究和防治产品开发的基础。信号标签诱变(signature-tagged mutagenesis,STM)是一种经过优化的经典的转座子突变技术,可以用含多个序列标签的转座子构建病原菌的突变体库,通过宿主或实验动物来负向筛选致弱突变体,进而鉴定相关基因。自1995年首次建立以来,STM技术被广泛用于多种病原菌毒力相关基因的发掘与鉴定。很多病原菌为了适应体内外的环境会形成生物被膜(biofilm)。大量的研究表明,生物被膜的形成与病原菌的免疫逃避和抗药性有关,是影响感染的重要因素。本研究构建了APP血清1型的STM突变体库,从中筛选到2个生物被膜形成突变菌株,对突变基因进行了定位和克隆,对突变菌株的生物学特性进行了研究,为阐明APP生物被膜的形成机制提供了一定的试验数据。1.APP血清1型STM突变体库的构建以APP血清1型萘啶酸抗性菌株4074-N为受体菌,以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E.coli CC118λpir或Sm17-1λpir为供体菌,在或不在E.coli DH5α(pRK2073)的辅助下,进行三亲本或两亲本接合,通过转座子内携带的卡那霉素抗性筛选、氨苄青霉素负筛选、PCR和Southern杂交鉴定转座突变株。在APP与E.coli接合实验中,通过对两亲本接合与三亲本接合进行了比较,证明了两亲本接合的效率明显高于三亲本接合。用两亲本接合方法,构建了32个STM标签的APP突变体库,共得到1652个转座突变菌株(每个标签不少于50个突变体)。2.APP生物被膜形成突变体的筛选及插入失活基因的鉴定用96孔板法和试管法从上述APP血清1型4074-N株(不产生生物被膜)STM突变体库中筛选到两株能产生很强生物被膜的突变体,即STM1-21和STM6-42。提取突变体的基因组用转座子内的SspI酶切后进行自连接。以连接产物为模板,用转座子上的特异性引物STM10/11和STM10/12,通过反向PCR扩增,获得转座子插入位点两侧的基因片段。经DNA测序和BLAST分析,发现这两株突变体均为Mini-Tn10转座子插入失活编码一种类组氨酸核结构蛋白(Histone-like nucleoidstructuring protein)的hns基因造成的。PCR和Southern杂交进一步确定了插入突变体的正确性。3.APP H-NS的结构与功能的初步研究APP的hns基因编码一种135个氨基酸的类核结构蛋白H-NS,主要由三个结构域组成,即N端的聚合结构域、C端的DNA结合结构域和中间的柔性连接片段。通过N端的聚合结构域形成多聚体结构,C端的DNA结合结构域与靶基因结合而发挥调控作用。为了研究APP的hns基因的功能,将4074-N株的hns基因表达盒(包括其编码区、启动子和终止子序列)克隆到能穿梭质粒pJN105中,构建了一个重组表达质粒pJN-hns,分别电转化到hns转座突变菌株STM1-21(M)和亲本菌株4074-N(P)中,期望获得互补菌株(C-3)和过表达菌株(0-2);然后比较分析了上述四个菌株P、M、C-3和O-2的生长特性、生物被膜形成和毒力的变化,结果表明:4个菌株的体外生长速度未见明显差异;除突变菌株M外,其它3个菌株均不能形成可测的生物被膜;菌株M、C-3和O-2对小鼠的毒力和溶血活性均有不同程度的减弱。互补菌株和过表达菌株的上述表型均与预期的结果不一致。为了进一步解释这种现象,我们通过实时荧光定量RT-PCR分析了上述4个菌株中hns基因及两种重要毒力基因axpⅠA和axpⅡA的表达情况。结果显示,hns基因在C-3和O-2菌株中均有表达,但表达量均比亲本菌株P还要低,提示H-NS的缺失在C-3株中并没有得到有效互补,H-NS也没有在O-2株中过表达,C-3和O-2均表现为hns的下调表达菌株,这可能是因为hns基因的表达存在自调控机制,这种表达自调控机制在大肠杆菌中已有报道。此外,axpⅠA和axpⅡA的表达下调也初步解释了菌株M、C-3和O-2的毒力与溶血活性减弱现象。为了进一步研究H-NS蛋白对APP生物被膜形成的影响,本实验以APP血清1型4074株基因组DNA为模板,PCR扩增了408 bp的hns基因编码区,克隆到原核表达载体pET-28c中获得重组质粒pET28c-hns,转化大肠杆菌(Escherichia coli)BL21(DE3),经IPTG诱导表达和组氨酸亲和层析柱纯化获得大小约19 kD的重组蛋白rH-NS。将不同浓度的rH-NS添加到hns突变株1-21及其亲本菌株4074的培养基中,用微孔板法测定生物被膜的形成。结果显示,在不添加rH-NS时,4074株不能形成可见的生物被膜,而1-21株形成明显的生物被膜;在添加0.1～0.3μmol/L的rH-NS的情况下,1-21株生物被膜的形成量随着rH-NS浓度的升高而降低,而4074株随着rH-NS浓度的升高而升高;rH-NS添加量超过0.4μmol/L对两个菌株生物被膜形成未见明显影响。结果表明H-NS蛋白负调控APP生物被膜的形成,并呈现一定的剂量效应。可见,H-NS作为一种DNA结合蛋白,不仅可以调控其它重要基因的表达,其自身的表达也受到严格的调控。它所调控的靶基因及自身调控机制有待进一步研究。更多还原

【Abstract】 Actinobacillus pleuropneumoniae(APP) is the causative agent of porcine contagious pleuropneumonia(PCP),a severe infectious respiratory disease of swine causing great economic losses to the pig industry worldwide.APP is obligatory parasitic bacteria of porcine respiratory,spreading through air and direct contact.Due to many distinct serotypes and lack of cross-protection among them,it is difficult to prevent and control the disease using traditional attenuated bacterins and/or subunit vaccines.So novel safe and effective vaccines are urgently needed to prevent and control this disease.The overall identification of virulence factors is the basis to understand the pathogenesis and develop new control products.Signature-tagged mutagenesis(STM) is an improved transposon mutagenesis technique.Using mini-transposon carrying different sequence tags,a series of transposal mutant pools could be constructed.Then attenuated mutant strains could be negatively selected from the mutant pools in host and/or experimental animals.Since the first application in 1995,STM is widely used to pick up the infecteion-associated genes in many kinds of bacteria.In order to adapt to the changed environments,many bacteria could form biofilms in vitro and in vivo.A plenty of evidence demonstrated that biofilm formation is involved in the immune escape and antibiotics resistance in many species of pathogenic bacteria.It plays an important role in the full infection and pathogenesis.In the present study,an STM mutant bank of APP serotype 1(a non-biofilm forming strain) was constructed.Two biofilm-forming mutant strains were identified from the mutant bank.The mutated gene was mapped,cloned and sequenced.Further characterization of the two biofilm-forming mutants provided useful experimental data to understand the mechanism of biofilm formation in this important pathogen.1.Constrcution of an STM mutant bank of APP serotype 1A nalidixic acid-resistant strain 4074-N of APP serotypes 1 was used as recipient strain to mate with E.coli CC 118λpir or Sm17-1λpir containing mini-Tn 10 tag plasmids pLOF/TAG1-48,with or without the help of E.coli DH5α(pRK2073).Mutant strains were screened by kanamycin resistance selection,ampicillin negative selection,PCR and Southern blot identification.In the mating experiments,the bi-parental mating was more effective and easy than tri-parental mating.Using the bi-parental mating method eatablished for APP,thirty two STM mutant pools have been constructed.Each pool contained at least 50 mutant strains. 2.Screening of biofilm-producing mutants and identification of inactivated genesUsing 96-well microtiter plate and glass tube assays,two strong biofilm-producing strains STM1-21 and STM6-42 were screened from the STM mutant pools described above.The genomic DNA of the mutants was extracted,digested with SspⅠlocated in the transposon,and self ligated.Using the ligated mixture as template,the both flank fragments of the transposon were amplified by reverse PCR with the transposon-specific primers STM10/11 and STM10/12 respectively.Sequence determination and analysis revealed that the hns gene encoding the histone-like nucleoid structuring protein(H-NS) was inactivated by the insertion of the mini-Tn10 transposon.Further PCR and Southern blots analysis confirmed the insertion.3.Structural and functional characterization of the hns gene in APPThe hns gene of APP encodes a histone-like nucleoid structuring protein(H-NS) consisting of 135 amino acids.The H-NS protein consists of an N-terminal dimerization domain and a C-terminal nucleic acid-binding domain that are separated by a linker region.It can form homodimer,tetramer or oligomer via its N-terminal dimerization domain,and the C-terminal nucleic acid-binding domain is important to bind DNA in regulation of gene expression.In order to understand the function of the hns gene in APP,the hns expression cassette,containing the complete coding sequence of hns and its native promoter and terminator,was amplified from the genomic DNA of APP strain 4074-N,and then cloned into the shuttle vector pJN105.To obtain a complementary strain and an over-expressing strain,the resultant expression plasmid pJN-hns was transformed into the transposal mutant strain STM1-21(M) and parental strain 4074-N(P) respectively,resulting in strain C-3 and 0-2.The in vitro growth property,biofilm formation and virulence were compared among the 4 strains P,M,C-3 and O-2.The results showed that no obvious difference in the in vitro growth was observed among the four strains.Except strain M forming obvious biofilms in vitro,no biofilm formation was visible in the other three strains.The virulence was attenuated in the strains M,C-3 and O-2 when their haemolytic activity and 50%lethal doses in mice were compared with the parental strain P.In order to explain the tmexpected phenotypes of strains C-3 and O-2,Real-time RT-PCR analysis was performed to compare the expression levels of hns gene and the two major virulence genes apxⅠA and apxⅡA.The results showed that the hns gene was in deed expressed in strains C-3 and O-2,but their levels were lower than that in the parental strain P.This indicated that the hns deletion was not completely complemented in strain C-3,and hns was not overexpressed in strain O-2 as well.Both strains C-3 and O-2 were actually hns knockdown strains.This may be due to the auto-regulation mechanism of hns expression which was reported in E.coli previously.H-NS may regulate the expression of target genes in a dose dependent manner,and the down-regulation of the exotoxin genes(apxⅠA and apxⅡA) in the hns deletion and knockdown strains may partly contribute to the virulence attenuation.To further investigate the effect of H-NS on APP biofilm formation,the 408 bp complete coding sequence of hns gene was amplified by PCR from the genomic DNA of APP serotype 1 strain 4074 and cloned into the prokaryotic expression vector pET-28c.The resultant recombinant plasmid pET28c-hns was transformed into Escherichia coli BL21(DE3).A 19 kD of recombinant protein(rH-NS) was obtained by IPTG induction,and purified using the Ni-NTA agarose columns.Different concentrations of rH-NS were added into the culture medium of the hns transposal mutant strain 1-21 and its parental strain 4074, and biofilms were quantified using the microtiter plate biofilm assay.Without rH-NS in the medium,strain 1-21 formed obvious biofims,but strain 4074 did not.Supplemented with 0.1～0.3μmol/L rH-NS in the culture medium,the biomass of biofilms formed by strain 1-21 continuously decreased,whereas that formed by strain 4074 was increased. No obvious changes could be observed when more than 0.4μmol/L of rH-NS was supplemented in both strains.Results indicated that H-NS negatively regulates biofilm formation of A.pleuropneumoniae in a dose-dependent manner.Our data indicate that H-NS plays important roles in regulating biofilm formation and virulence in A. pleuropneumoniae.The H-NS regulated genes and regulatory mechanism need futher investigation.更多还原