The adherence of Bordetela pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract.Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin(Fha).The carbohydrate recognition domain(CRD)of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization.For identification of the CRD of Fha,a bank of eight monoclonal antibodies(MAbs)that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells.Only MAb 12.5A9,which maps to amino acid residues 1141 to 1279,blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells.An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli.Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha.Mutant strains of B.pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9.In an adherence assay,these mutant strains failed to bind efficiently to either ciliated cells or macrophages.The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain.We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine.

Although pneumococcus is one of the most frequently encountered opportunistic pathogen in the world,the mechanisms responsible for its infectiveness have not yet been fully understood.In this paper,we have attempted to characterize the effects of pneumococcal transformation on the pathogenesis of the organism.We constructed three transformation-deficient pneumococcal strains,which were designated as Nos.1d,2d,and 22d.The construction of these altered strains was achieved via the insertion of the inactivated gene,comE,to strains 1,2 and 22.We then conducted a comparison between the virulence of the transformation-deficient strains and that of the wild-type strains,via an evaluation of the ability of each strain to adhere to endothelial cells,and also assessed psaA mRNA expression,and the survival of hosts after bacterial challenge.Compared to what was observed with the wild-type strains,our results indicated that the ability of all of the transformation-deficient strains to adhere to the ECV304 cells had been significantly reduced (p<0.05),the expression of psaA mRNA was reduced ignificantly (p<0.05) in strains 2d and 22d,and the median survival time of mice infected with strains 1d and 2d was increased significantly after intraperitoneal bacterial challenge (p<0.05).The results of our study also clearly indicated that transformation exerts significant effects on the virulence characteristics of S.pneumoniae,although the degree to which this effect is noted appears to depend primarily on the genetic background of the bacteria.

用F-actin特异性FITC-phalloidin荧光染料，观察肺炎链球菌（StrepLococcus penumonioe）作用A549细胞前后的F-actin细胞骨架重排情况；用细胞松弛素D预处理A549细胞，观察肺炎链球菌对A549细胞的侵袭率；使用Datrolene预处理A549细胞，观察其与F-actin细胞骨架重排百分率间是否存在剂量依赖关系；用Fura-2/AM荧光探针负载A549细胞后测定肺炎链球菌粘附A549细胞后的胞内Ca2+浓度.结果发现肺炎链球菌作用A549细胞后，F-actin细胞骨架呈块状、丝状聚集；而松弛素D可明显降低肺炎链球菌对A549细胞的侵袭率；肺炎链球菌粘附A549细胞后胞内Ca2+高于对照；Datrolene可部分抑制A549细胞F-actin细胞骨架重排，且与Factin细胞骨架重排百分率间存在量效关系。以上结果提示肺炎链球菌可通过Ca2+细胞信号转导途径触发A549细胞Factin细胞骨架重排，进而导致肺炎链球菌侵袭A549细胞：

目的通过体外实验诱导的转化过程，探讨肺炎链球菌（Streptococcus pneumoniae，S.pn）毒力因子的表达是否与环境诱导细菌感受态形成与转化有关。方法采用插入失活的方式断裂S.pn感受态形成的关键基因comE，获得感受态缺陷菌株。以实时定量RT-PCR测定毒力因子在感受态缺陷菌与野生菌的表达是否存在差异。所测毒力因子有自溶酶、溶血素、胆碱结合蛋白、S-IgA、神经氨酸酶、透明质酸酶。结果 S.pn在540nm处吸光度（A）值=0.044 -0.127之间产生感受态。自溶酶、神经氨酸酶、透明质酸酶3种毒力因子的表达，野生菌组表达高丁缺陷菌组，两均数间比较的t值分别为2.96（P<0.05）、2.8（P<0.05）、4.56（P<0.05）。结论 细菌感受态能启动自溶酶、神经氨酸酶、透明质酸酶3种毒力因子的表达，提示S.pn的感受态能影响其毒力因子的表达。

目的通过体外实验研究肺炎链球菌（Streptococcus pnezunoniae，S.pn）侵袭A549细胞和微丝肌动蛋白细胞骨架重排之间的关系。方法采用Factin特异性FITC-phalloidin荧光染料，观察_s.pn作用于A549细胞前后的F—actin细胞骨架重排情况；用F—actin细胞骨架重排抑制剂——细胞松弛素D预处理A549细胞，观察S.pn对A549细胞的侵袭率；分别使用PLC、TPK信号转导抑制剂U73122、（enistein处理因素预处理A549细胞，观察其与F—actin细胞骨架重排的关系。结果S.pn作用4，49细胞后，F-actin细胞骨架呈块状、丝状聚集；F-actin细胞骨架重排抑制剂细胞松弛素D可明显降低s.pn对A549细胞的侵袭率，在其浓度为0.25ug/ml时，未得到可测的细菌数；PLC（磷脂酶C）、TPK（酪氨酸蛋白激酶）信号转导途径抑制剂可部分抑制4 549细胞Factin细胞骨架重排，其相关系数分别为：rPLC=-0.88（P<0.05）； rTPK=-0.91（P<0.05）。S.pn细胞壁作用4549细胞后，Factin绌胞骨架呈块状、丝状聚集，二者间存在剂量依赖关系。结论S.pn可通过PLC、TPK细胞信号转导途径触发A549细胞F-actin细胞骨架重排，进而导致S.pn侵袭A549细胞

分析肺炎链球菌细胞壁胆碱成分在其侵袭宿主细胞的过程中的作用。通过肺炎链球菌对人脐静脉内皮细胞的侵袭实验，观察血小板活化因子受体（PAF-R）拮抗剂BN 52021对肺炎链球菌侵袭率的变化，以及乙醇胺取代细胞壁胆碱后肺炎链球菌侵袭率的变化。研究发现受体拮抗剂BN 52021处理活化血管内皮细胞后，肺炎链球菌的侵袭率显著降低（P<0.01），乙醇胺取代肺炎链球菌细胞壁成份中的胆碱同样降低了细菌对活化内皮细胞的侵袭（P<0.01）。实验表明肺炎链球菌可能是通过脂磷壁酸和磷壁酸的胆碱成分与内皮细胞表面的血小板活化因子受体结合来介导侵袭作用的。

目的探讨肺炎链球菌在对抗p内酰胺类抗生素时是否受感受态形成的影响。方法通过转化得到感受态缺陷菌株，采用半定量RT-PCR检测相关基因ciaH和cpaA在细菌感受态形成时的表达，用肉汤法检测各菌株的最低抑菌浓度（MIC）。结果细菌感受态时基因ciaH和cpaA的mRNA表达量增高（P<0.05），野生菌感受态缺陷后的rvnc值发生改变。结论肺炎链球菌的感受态形成可诱导ciaH和cpoA的表达增加，其感受态缺陷影响到其对p内酰胺类抗生素的敏感性，但不同菌株的影响不同。

以绿色荧光蛋白（GFP）作为体内研究的分子探针，将肺炎链球菌毒力基因与幽融合构建肺炎链球菌自杀性荧光报告质粒，利用同源重组的原理，使gfp整合入肺炎链球菌基因组中，建立体内研究肺炎链球菌基因表达的荧光报告系统，并用荧光激发、生物学特征和生理活性测定等实验手段进行评价，证实这一肺炎链球菌荧光融合表达系统可在体内外报告肺炎链球菌的毒力基因表达，为进一步在体内分析和鉴定肺炎链球菌毒力因子的功能奠定基础。

目的探讨肺炎链球菌的自然转化与其条件致病的关系。方法采用插入失活的方法制备肺炎链球菌的转化缺陷菌株，通过黏附实验和小鼠毒力实验观察它们毒力的变化，应用RT-PCR 测定黏附因子PsaA在野生和缺陷菌株中的表达。结果实验获得了3株肺炎链球菌（1、2和22）的转化缺陷菌株（1d、2d和22d）。毒力研究发现转化缺陷菌株对ECV 304 细胞的黏附能力显著弱于相应的野生菌株，1和2 株肺炎链球菌腹腔感染BALB/c小鼠的能力显著强于相应的转化缺陷菌株；研究还发现 PsaA 基因在2 和22 株肺炎链球菌中的表达量显著高于其缺陷菌株。结论肺炎链球菌具有自然转化的能力有助于其毒力的表现，且可能通过黏附因子 PsaA 等相关毒力因子表达增加而增强其毒力。