Ⅴ型分泌系统菌体表面展示F18大肠杆菌黏附素及其受体结合位点的确定

自主转运蛋白(V型分泌系统)的β结构域已被证明可以将异源性多肽展示在细菌表面。运用DNA重组技术优化构建V型分泌系统MisL并在菌体表面展示F18大肠杆菌黏附素FedF及其受体结合域FedF1。含重组质粒pnirBMisL-fedF或pnirBMisL-fedF1大肠杆菌(E.coli)DH5α经厌氧诱导后,分别与兔抗F18ab菌毛FedF亚单位单因子血清和F18大肠杆菌黏附素受体易感性仔猪的小肠上皮细胞做玻板凝集试验和体外黏附试验,结果表明上述两株诱导表达重组菌与FedF抗血清发生明显的凝集反应,且能较好地黏附于F18大肠杆菌黏附素受体易感性仔猪小肠上皮细胞。而菌体表面展示F18大肠杆菌黏附素FedF突变体FedF(M)(黏附素受体结合域第88和89位组氨酸残基双突变为丙氨酸)的重组菌则失去上述凝集和黏附特性。以上试验结果说明,F18大肠杆菌黏附素FedF及其受体结合域FedF1在大肠杆菌表面得到了功能性表达,并进一步证明了位于FedF受体结合域内第88和89位组氨酸残基对FedF受体结合域的形成至关重要。     

大肠杆菌F4在3个品种猪中的黏附模式

大肠杆菌F4是引起仔猪断奶前腹泻的一种主要细菌,F4黏附于小肠上皮细胞是其致病的前提。小肠上皮细胞的F4受体是由常染色体上的基因编码的,如果无受体,仔猪表现为大肠杆菌抗性。为了研究黏附的遗传机制,本实验利用大白、长白、松辽黑猪的小肠刷状缘细胞与F4ab、F4ac、F4ad进行离体黏附实验,结果发现3品种(系)猪之间黏附情况存在显著差别(P<0.01),松辽黑猪以非黏附型为主,而长白猪中黏附型比例较高,在同一品种猪内,3种菌株的黏附比例在松辽黑猪内和大白猪内有极显著差异,但在长白猪中无显著差异。从3种细菌与刷状缘的黏附模式来看,F4ab、F4ac和F4ad分别有3种不同的受体,它们可能是由3个不同的基因座编码的。     

大肠杆菌K88ac菌毛操纵子fae全基因的克隆、表达及生物学活性初步研究

目的：在体外克隆和表达猪肠产毒性大肠杆菌（ETEC）K88ae菌毛操纵子,触结构基因,并检测重组菌毛的相关生物学活性。方法：利用长PCR技术以猪ETECK88ae株C83902基因组DNA为模板扩增编码K88菌毛操纵子触基因,克隆入表达质粒载体pBR322,构建和筛选重组质粒pBR322-fae,转化至不含任何菌毛的大肠杆菌EP株;电镜观察重组菌表面菌毛表达情况;用热抽提法提纯表达的重组菌毛;用纯化菌毛免疫小鼠制备高效价抗血清;用SDS-PAGE和Western blot检测重组菌毛的抗原性,用细胞黏附和黏附抑制试验检测其生物学活性。结果和结论：在电镜下观察到重组菌表面大量表达K88ae菌毛,该重组菌与兔抗K88ae菌毛单因子阳性血清、鼠抗K88ac菌毛单克隆抗体均产生凝集反应;纯化菌毛经SDS-PAGE,结构单位菌毛呈单一的相对分子质量约26×10^3的蛋白条带;纯化菌毛免疫小鼠后可制备出高效价的鼠抗血清,玻板凝集试验和Western blot结果表明体外表达的K88ae菌毛具有与K88ae野生菌毛相同的抗原性;猪小肠上皮细胞系黏附和黏附抑制实验结果表明重组EP菌和野生菌株一样具有较强的黏附猪小肠上皮细胞系的能力,而且提纯重组菌毛制备出的鼠抗血清能有效抑制上述重组菌或野生菌株对猪小肠上皮细胞系的黏附结合。     

大肠杆菌F18菌毛操纵子全基因克隆、表达及生物学活性

利用PCR技术以猪产肠毒素大肠杆菌F18标准菌株107/86和2134P基因组DNA为模板成功地扩增出编码F18ab和F18ac完整菌毛操纵子fed基因。将它们分别克隆入表达质粒载体pET-22b( ),结合酶切和核苷酸序列分析证明了PCR预期扩增产物的正确性。然后将克隆的重组载体DNA转化至大肠杆菌BL21(DE3),构建和筛选出分别含F18ab和F18ac完整fed基因的重组菌,经过IPTG诱导表达,在电镜下观察到上述两种重组菌能分别大量表达F18ab和F18ac菌毛。用热抽提法提纯其诱导表达的F18ab和F18ac菌毛,经SDS-PAGE电泳和考马斯亮蓝染色发现提纯后菌毛获单一分子量约为15kDa蛋白条带,免疫家兔后制备出高效价的兔抗血清,玻板凝集试验和Western blot结果表明:体外诱导表达的F18ab和F18ac菌毛具有和野生F18菌毛相同的抗原性。用表达F18ab和F18ac菌毛的上述2株重组菌分别进行小肠上皮细胞体外吸附试验和吸附抑制试验,结果表明:2株重组菌和野生菌株一样具有较强的粘附易感仔猪小肠上皮细胞的能力,而用表达F18ab和F18ac重组菌提纯的菌毛制备出兔抗血清都能有效地抑制上述重组菌或野生菌株对易感仔猪小肠上皮细胞的吸附结合。     

动物源产肠毒素大肠杆菌(ETEC)黏附素研究进展

动物源产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)是引起动物(尤其是幼龄动物)腹泻的主要病原菌。已知黏附素和肠毒素是ETEC中两种重要的毒力因子,在致病性中两者缺一不可。其中黏附素结合到宿主易感肠上皮细胞是ETEC感染的第一步,也是最重要的关键步骤。动物源ETEC的菌毛黏附素主要包括K88、K99、987P、F18、F17和F41等。人们从20世纪60年代就开始了ETEC菌毛黏附素的相关研究,包括菌毛的基因、结构组成、生物合成、菌毛表达的调控机制以及黏附素和宿主受体相互作用等,这些研究基础有助于我们深入了解ETEC病原菌的感染机理;并且在疾病诊断和新疫苗的开发中具有重大意义。     

肺炎球菌表面蛋白的克隆表达与免疫原性研究

从肺炎球菌YF05中扩增了肺炎球菌表面蛋白A（PspA）和肺炎球菌表面黏附素A（PsaA）基因,以pET27b(+)为载体构建了重组表达质粒的表达系统后,转化入大肠杆菌BL21,IPTG诱导表达,表达的重组蛋白约占菌体总蛋白的75％,结果显示：表达的PspA蛋白和PsaA蛋白,分子量分别约为75kDa和37kDa。成功表达的重组蛋白具有较强的免疫活性和交叉免疫效果。     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

猪产肠毒素大肠杆菌987P蛋白受体的鉴定

产肠毒素大肠杆菌(ETEC)定植于仔猪肠道的第一步是通过987P菌毛与小肠上皮细胞表面刷状缘大分子(BBV)结合。对分离的BBV进行SDS-PAGE和Ligand blot分析表明, 在32~35KDa区域内有一条带能被987P菌毛探针所识别和结合, 所结合的条带经胰蛋白酶消化后, 通过微内径反相高效液相色谱(RP-HPLC)分离出多条主要峰带蛋白峰带, 采用衬质辅助激光解吸与电离质谱法(MALDI-MS)对主要峰带进行分析, 结合多肽氨基酸测序和Blast同源性比较, 得到3个氨基酸基序(AETAP、ALAAAGYDVEK和LGLK), 其序列与人和鼠源的组蛋白H1高度同源; 来源于仔猪小肠上皮细胞BBV的H1蛋白与BBV一样都能特异性结合纯化的987P菌毛蛋白。上述结果表明, 仔猪小肠上皮细胞BBV的组蛋白H1是987P菌毛蛋白的受体。     

基于大肠杆菌受体菌DH5α △asd缺失株的构建及作为基因转化、表达操作系统的评估

基于大肠杆菌(E.coli)染色体上asd基因的已知序列,利用λ噬菌体的Red同源重组系统一步法构建E.coliDH5α的asd基因缺失突变株DH5α△asd::cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20介导二次同源重组,以去除上述缺失突变株中氯霉素抗性筛选基因。结合PCR扩增和测序结果,证明DH5α△asd缺失突变株的正确构建。该缺失突变株失去了在普通LB培养基上生长的能力,只有添加DAP或导入表达asd基因的质粒(asd基因互补试验)才能在LB培养基上生长,与原型DH5α比较,其生长速度和生长对数期、接受不同拷贝数质粒的转化效率几乎相一致。基于该缺失突变株构建出以asd营养基因为标志的大肠杆菌染色体-质粒平衡致死系统。体外培养连续传代50代次,pnirBMisL-fedF-asd质粒不丢失,并功能性表达F18大肠杆菌黏附素FedF。     

禽致病性大肠杆菌Ⅰ型菌毛fimH基因缺失突变株的构建 及部分生物学特性的分析

基于禽大肠杆菌Ⅰ型菌毛黏附素fimH基因的已知序列,利用入噬菌体的Red重组系统构建禽致病性大肠杆菌国内分离株A2（血清型O2：K89）Ⅰ型菌毛黏附素fimH基因缺失突变株A2△fimH：：Cat,在二次重组中利用携带能够表达FLP位点特异性重组酶的质粒pCP20（温度敏感性）以去除上述缺失突变株中抗性基因标志,结合PCR扩增和测序结果,证明fimH基因缺失株A2AfimH的正确构建。通过fimH基因互补试验使A2afimH缺失突变株恢复了与野生株具有相同的凝集活性。红细胞和酵母细胞凝集试验结果表明,野生株呈现良好的凝集效果,并能被0．5％甘露糖完全抑制,而A2afimH缺失突变株未呈现任何凝集现象。体外生长试验结果表明,在同样的培养条件下,A2afimH缺失突变株生长周期的各个阶段都要稍慢于野生株。禽致病性大肠杆菌国内分离株Ⅰ型菌毛黏附素fimH基因缺失突变株成功构建,为进一步深入研究禽大肠杆菌Ⅰ型菌毛与机体相互作用的分子机制,肠道外感染的致病机理及对国内禽大肠杆菌病的防控策略奠定了一定基础。     

Structure of the Y. pseudotuberculosis adhesin InvasinE

Enteropathogenic Yersinia expresses several invasins that are fundamental virulence factors required for adherence and colonization of tissues in the host. Within the invasin‐family of Yersinia adhesins, to date only Invasin has been extensively studied at both structural and functional levels. In this work, we structurally characterize the recently identified inverse autotransporter InvasinE from Yersinia pseudotuberculosis (formerly InvasinD from Yersinia pseudotuberculosis strain IP31758) that belongs to the invasin‐family of proteins. The sequence of the C‐terminal adhesion domain of InvasinE differs significantly from that of other members of the Yersinia invasin‐family and its detailed cellular and molecular function remains elusive. In this work, we present the 1.7 Å crystal structure of the adhesion domain of InvasinE along with two Immunoglobulin‐like domains. The structure reveals a rod shaped architecture, confirmed by small angle X‐ray scattering in solution. The adhesion domain exhibits strong structural similarities to the C‐type lectin‐like domain of Yersinia pseudotuberculosis Invasin and enteropathogenic/enterohemorrhagic E. coli Intimin. However, despite the overall structural similarity, the C‐type lectin‐like domain in InvasinE lacks motifs required for Ca²⁺/carbohydrate binding as well as sequence or structural features critical for Tir binding in Intimin and β₁‐integrin binding in Invasin, suggesting that InvasinE targets a distinct, yet unidentified molecule on the host‐cell surface. Although the biological role and target molecule of InvasinE remain to be elucidated, our structural data provide novel insights into the architecture of invasin‐family proteins and a platform for further studies towards unraveling the function of InvasinE in the context of infection and host colonization.     

Structure of the head of the Bartonella adhesin BadA

Trimeric autotransporter adhesins (TAAs) are a major class of proteins by which pathogenic proteobacteria adhere to their hosts. Prominent examples include Yersinia YadA, Haemophilus Hia and Hsf, Moraxella UspA1 and A2, and Neisseria NadA. TAAs also occur in symbiotic and environmental species and presumably represent a general solution to the problem of adhesion in proteobacteria. The general structure of TAAs follows a head-stalk-anchor architecture, where the heads are the primary mediators of attachment and autoagglutination. In the major adhesin of Bartonella henselae, BadA, the head consists of three domains, the N-terminal of which shows strong sequence similarity to the head of Yersinia YadA. The two other domains were not recognizably similar to any protein of known structure. We therefore determined their crystal structure to a resolution of 1.1 A. Both domains are beta-prisms, the N-terminal one formed by interleaved, five-stranded beta-meanders parallel to the trimer axis and the C-terminal one by five-stranded beta-meanders orthogonal to the axis. Despite the absence of statistically significant sequence similarity, the two domains are structurally similar to domains from Haemophilus Hia, albeit in permuted order. Thus, the BadA head appears to be a chimera of domains seen in two other TAAs, YadA and Hia, highlighting the combinatorial evolutionary strategy taken by pathogens.     

Structure refinement of the OpcA adhesin using molecular dynamics

OpcA from Neisseria meningitidis, the causative agent of meningococcal meningitis and septicemia, is an integral outer membrane protein that facilitates meningococcal adhesion through binding the proteoglycan receptors of susceptible cells. Two structures of OpcA have been determined by x-ray diffraction to 2 A resolution, revealing dramatically different conformations in the extracellular loops--the protein domain implicated in proteoglycan binding. In the first structure, a positively charged crevice formed by loops 1 and 2 was identified as the site for binding proteoglycans, whereas in the second structure the crevice was not evident as loops 1 and 2 adopted different conformations. To reconcile these results, molecular-dynamics simulations were carried out on both structures embedded in a solvated lipid bilayer membrane. Free of crystal contacts and crystallization agents, the loops were observed to undergo large structural transformations, suggesting that the conformation of the loops in either x-ray structure is affected by crystallization. Subsequent simulations of both structures in their crystal lattices confirmed this conclusion. Based on our molecular-dynamics trajectories, we propose a model for OpcA that combines stable structural features of the available x-ray structures. In this model, all five extracellular loops of OpcA have stable secondary structures. The loops form a funnel that leads to the base of the beta-barrel and that includes Tyr-169 on its exposed surface, which has been implicated in proteoglycan binding.     

Analysis of genes coding for the sialic acid-binding adhesin and two other minor fimbrial subunits of the S-fimbrial adhesin determinant of Escherichia coli

The S fimbrial adhesin (Sfa) enables Escherichia coli to attach to sialic acid-containing receptor molecules of eukaryotic cells. As previously reported, the genetic determinant coding for the Sfa of an E. coli O6 strain was cloned, the gene coding for the major fimbrial subunit was identified and sequenced and the S specific adhesin was detected. Here we present evidence that in addition to the major subunit protein SfaA three other minor subunit proteins, SfaG (17 kD), SfaS (14 kD) and SfaH (31 kD) can be isolated from the S-specific fimbrial adhesin complex. The genes coding for these minor subunits were identified, mutagenized separately and sequenced. Using haemagglutination tests, electron-microscopy and quantitative ELISA assays with monoclonal anti-SfaA and anti-SfaS antibodies the functions of the minor subunits were determined. It was determined that SfaS is identical to the S-specific adhesin, which also plays a role in determination of the degree of fimbriation of the cell. The minor subunit SfaH also had some influence on the level of fimbriation of the cell, while SfaG is necessary for full expression of S-specific binding. It was further shown that the amino-terminal protein sequence of the isolated SfaS protein was identical to the protein sequence calculated from the DNA sequence of the sfaS gene locus.     

Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.     

The role of adhesin in the polymeric structure of Vi-antigen

Samples of Vi-antigen subjected to hydrolysis of different duration have been studied. As revealed in this study, 12-hour hydrolysis causes the depolymerization of the preparation. The fragments thus obtained have been found to possess Vi-specificity. The polymeric form of Vi-antigen contains adhesin differing in specificity from Vi-determinant. This component is absent in the low-molecular fraction of Vi-antigen. Adhesin is the binding element of the molecule of Vi-antigen and ensures its polymeric structure.     

Functional organization of the autotransporter adhesin involved in diffuse adherence

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. To elucidate the structure-function relationships of AIDA-I, we performed transposon-based linker scanning mutagenesis and constructed mutants with site-directed deletions. Twenty-nine different mutants with insertions that did not affect protein expression were obtained. Eleven mutants were deficient for one or two but not all of the functions associated with the expression of AIDA-I. Functional characterization of the transposon mutants and of an additional deletion mutant suggested that the N-terminal third of mature AIDA-I is involved in binding of this protein to cultured epithelial cells. The purified product of the putative domain could bind to cultured epithelial cells, confirming the importance of this region in adhesion. We also identified several different mutants in which invasion and adhesion were changed to different extents and two mutants in which autoaggregation and biofilm formation were also affected differently. These results suggest that although conceptually linked, adhesion and invasion, as well as autoaggregation and biofilm formation, are phenomena that may rely on distinct mechanisms when they are mediated by AIDA-I. This study sheds new light on the workings of a protein belonging to an emerging family of strikingly versatile virulence factors.     

Adherence epitopes of Mycoplasma genitalium adhesin.

The adherence-mediating sites of the 153 kDa adhesin of Mycoplasma genitalium (MgPa-protein) were characterized at the amino acid sequence level using six monoclonal anti-MgPa antibodies which showed adherence-inhibiting activity. For characterization of the regions to which antibody bound, three segments of the adhesin (N-terminal region, a D1-domain located approximately in the middle of the molecule and a D2-domain located near to the C-terminus) were synthesized as overlapping octapeptides. These regions were chosen in analogy to the three domains of Mycoplasma pneumoniae that are involved in the adhesion process. Whereas two monoclonal antibodies (mAb 5B11 and mAb 6F3) bound exclusively to an epitope in the N-region, mAb 3B7 and mAb 6A2 reacted with two distinct epitopes of the D2-domain only. Binding to short synthetic peptides of different regions was analysed for mAb 3A12 (N-region and D1-region) and mAb 2B6 (N-region and D2-region). Close proximity of the N-region and the D2-region in the native MgPa-protein of M. genitalium was indicated in a competitive ELISA test, using freshly harvested M. genitalium cells. Epitope mapping and competition experiments with monoclonal anti-MgPa antibodies revealed interesting differences in the adherence-mediating sites of MgPa and the adhesin (P1-protein) of M. pneumoniae. Whereas a three-dimensional arrangement of protein loops is suggested for both native adhesins, the MgPa-protein and the P1-protein adherence-mediating epitopes are located in non-homologous regions of these two related proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

The amino-terminal domain of the P-pilus adhesin determines receptor specificity

Pyelonephritic isolates of Escherichia coli commonly express P-pili, which mediate bacterial attachment to glycolipids on epithelial cell surfaces. Three classes of P-pili have been defined, based on varying specificity for galabiose-containing glycolipids. Variation in adhesive capacity is correlated with a shift in preferred host, suggesting that host tropism depends largely on detailed specificity for the globoseries glycolipids. In this study we examined the importance of the PapG adhesin in determining receptor specificity. Translational fusions were constructed between the ammo-terminus of the PapG adhesin from each of the three pilus classes and a reporter protein. The binding specificity of the purified fusion proteins in vitro was identical to that seen with whole bacteria. Adherence of intact bacteria to cultured kidney cells was markedly reduced by a monoclonal antibody specific for the Class III adhesin (previously denoted PrsG), confirming the importance of the ammo-terminus of PapG in mediating attachment to a receptor when presented on the eukaryotic cell surface. These results suggest that the detailed receptor specificity resides solely within the amino-terminus of the PapG adhesin and is independent of the complex pilus architecture.