探讨dacA基因对化脓性链球菌感染过程的影响 盘尼西林结合蛋白 ; ...

探讨dacA基因对化脓性链球菌感染过程的影响 盘尼西林结合蛋白 ; ... 出版年: 研究生: 鄒志成 研究生(英文Chih-Cheng Tsou 姓名): 論文名稱: 探討 dacA基因對化膿性鏈球菌感染過程的影響 英文論文名Effect of the dacA gene in Streptococcus pyogenes infection 稱: 指導教授: 吳俊忠 指導教授(英Jiunn-Jong Wu 文姓名): 學位類別: 碩士 校院名稱: 國立成功大學 系所名稱: 微生物及免疫學研究所 學年度: 92 語文別: 中文 論文頁數: 99 關鍵詞: 盤尼西林結合蛋白 ; 化膿性鏈球菌 英文關鍵詞: penicillin-binding protein ; Streptococcus pyogenes 被引用次數: 0 [ 摘要 ] 化膿性鏈球菌為人類常見的致病菌,在臨床上引起的人類疾病從輕微的咽喉炎、皮膚感染到嚴重的壞死性肌膜炎、鏈球菌毒性休克症候群等。雖然已知化膿性鏈球菌感染需要許多的致病因子參與其中,但是真正的細菌致病分子機轉仍未被了解透徹。盤尼西林結合蛋白:PBPs:在革蘭氏陽性菌中不僅被發現參與細胞壁合成,亦發現在許多的動物感染模式中具有致病角色。化膿性鏈球菌的dacA基因位於opp 操縱子上游推測可能為盤尼西林結合蛋白,本論文主要探討dacA基因是否為盤尼西林結合蛋白以及此蛋白是否扮演在化膿性鏈球菌致病的角色。首先,我們利用插入法將dacA基因進行突變,以南方點墨法與PCR確認突變株。野生株與突變株的生長曲線並沒有顯著的差異,利用RT-PCR分析下游基因的表現發現突變dacA基因並無影響下游基因。在掃瞄氏電子顯微鏡下觀察兩者的型態也並無明顯差異性,然而在穿透式電子顯微鏡負染色下觀察,突變 株的細胞壁可能較為薄弱而有皺折的型態。野生株與突變株兩者的盤尼西林最小 抑制濃度皆小於0.06 ,慊/ml。利用螢光標記盤尼西林測試DacA是否具有盤尼 西林結合的角色,結果顯示dacA突變株造成PBP 4及PBP 5對盤尼西林親和 力的降低,推測DacA具有盤尼西林結合的功能。由脫脂牛奶培養基實驗得知 DacA突變株不會影響SpeB的表現,然而dacA突變株在細胞附著及細胞侵入 能力皆只有野生株的25%。將野生株與突變株同時與全血培養3小時比較兩者 的抗吞噬能力,結果顯示突變株的抗吞噬能力約只有野生株的十分之一。從 RT-PCR及莢膜分析發現突變株並不會影響莢膜與M protein的生成。再以 2×108 CFU菌數感染BALB/c小鼠,野生株在感染後兩天內所有的小鼠全部死 亡,而突變株感染小鼠後到第十天的存活率仍高達91,。綜合以上in vitro 和in vivo的結果,本研究證明DacA不僅具有盤尼西林結合能力,在化膿性鏈球菌感 染的過程中也扮演很重要的角色。未來仍須進一步探討DacA的PBP功能以及 以互補實驗證實dacA在化膿性鏈球菌的角色。 寡胜肽膜透酶由五個蛋白組成,附著於細胞膜上屬於ABC-transporter群。 先前的研究指出oppD和oppF突變會影響SpeB的表現,從氨基酸序列推測 OppD和OppF可能是具有ATPase活性之細胞膜內附著蛋白。先前本實驗室的 研究發現Opp具有雙重調節致病因子的角色,因此本研究擬進一步探討Opp是 直接或間接活化蛋白進而調控其致病基因表現。首先將oppF基因構築於 pET-21b表現載體,利用鎳離子親和管柱成功的得到重組r-OppF蛋白並經由西 方點墨法確認。採用非放射性實驗測定r-OppF的ATPase活性,發現r-OppF 水解ATP能力是隨時間和蛋白濃度比例增加。確認r-OppF具有ATPase活性 後,利用免疫沈澱法也發現許多蛋白與OppF結合,其中四個蛋白經由蛋白質身 份鑑定,其中一個蛋白推測為peroxide resistance protein。然而這些蛋白是如 何與OppF結合以及這些蛋白是否會調控其他致病基因的表現仍須日後深入研 究。 [ 英文摘要 ] Streptococcus pyogenes (Group A streptococcus, GAS) is the human pathogen that causes a variety of human diseases ranging from mild sore throat and skin infections to life-threatening necrotizing fasciitis and toxic shock syndrome. Although there are many virulence factors that have shown to be involved in GAS infection, the actual molecular mechanism of bacterial pathogenesis in GAS is far from clear. Penicillin-binding proteins (PBPs) in Gram-positive bacteria have not only shown to be involved in cell wall synthesis but also serve as a potential virulence factor in various animal models. Since the dacA gene of GAS is encoded with a putative penicillin-binding protein which is localized in the upstream region of the opp operon, we tested whether dacA has roles to serve as a penicillin-binding protein and as a virulence factor in GAS infection. First, we constructed an isogenic mutant by using integrational mutagenesis to disrupt the dacA gene and confirmed this by Southern blot and PCR analyses. The growth curves were not different between the wild-type strain and mutant. No polar effect was detected in the downstream of dacA by RT-PCR analysis. No difference of cell morphology was observed between mutant and wild-type strain using the scanning electronic microscope. When using the transmission electronic microscope, the mutant showed the pleat in the cell wall indicating it may be a thin cell wall. The minimal inhibitory concentration of penicillin in mutant and wild-type strain was less than 0.06 ug/ml. Second, we used fluorescent labeled penicillin to test whether DacA has a role as a penicillin-binding protein. The results showed that dacA mutant decreased the PBP4 and PBP5 affinity suggesting the DacA has a function as a penicillin-binding protein. Third, the dacA mutant did not affect SpeB expression that was analyzed by skim milk assay. However, the adhesion and invasion abilities of dacA mutant were about 75% less than those of the wild-type strain. When wild-type strain and dacA mutant incubated with heparinized whole blood for 3 h, the results demonstrated that the mutant had ten-fold decrease in antiphagocytosis activity than that of the wild-type strain. The RT-PCR analysis and hyaluronic acid measurement showed that the dacA mutant did not affect capsule and M protein expression. BAL/C mice were challenged with 2 × 108 CFU and results showed that mice infected with wild-type strain had 100% mortality after two days whereas those infected with dacA mutant had only 9% mortality after 10 days infection. Based on the in vitro and in vivo data, we demonstrate that DacA not only play a role as a penicillin-binding protein, but it also plays a role in bacterial virulence which contributes to the group A streptococcal infection. Further studies are needed to determine why DacA has a function as a PBP and verified the role of DacA in GAS by complementation assay. Oligopeptide permease (Opp) is a membrane-associated complex of five proteins (OppABCDF) belonging to the ABC-transporter family. Previous studies have demonstrated that mutations in oppD and oppF decrease the expression of SpeB. Analysis of the deduced amino acid sequences of the OppD and OppF indicated that it is a putative inner membrane associated with ATPase. Since we have found the Opp operon plays a dual role in regulation of virulence factors, we wonder if OppF may directly or indirectly activate responder proteins and regulate downstream of virulence factors. The oppF gene was cloned into pET-21b expression vector and r-OppF was expressed successfully. The r-OppF protein was purified by Ni2+-chelating column and confirmed by Western-blot analysis. We further tested whether r-OppF had the ATPase activity by non-isotope method and results revealed that r-OppF had ATPase activity. The ATPase activity was increased proportionally when protein concentrations and incubation times were increased. There were several proteins that interacted with r-OppF by immunoprecipitation method and four proteins were further identified. Among them, one protein was identified as a putative peroxide resistance protein. How this protein interacts with OppF and whether this protein has any effect on any virulence factors require further studies.