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創傷弧菌莢膜多醣體對生物膜形成及群體數量感應調控子LuxR/SmcR表現影響之研究

Studies on the Effect of Capsular Polysaccharides on Formation of Biofilm and Expression of Quorum-sensing Regulators LuxR/SmcR in Vibrio vulnificus

作者:吳昕玹
畢業學校:輔英科技大學
出版單位:輔英科技大學
核准日期:2013-01-29
類型:Electronic Thesis or Dissertation
權限:Copyright information available at source archive--Fooyin University....

中文摘要

創傷弧菌是一種嗜鹽性革蘭氏陰性桿菌,在自然環境中創傷弧菌存在於海水裏。創傷弧菌會造成人類的伺機性感染,感染包括敗血症與傷口感染。由於莢膜多醣體為潛在的毒力因子之一,與生物膜的形成有關。為了要探討莢膜多醣體對於創傷弧菌生物膜形成的影響,因此本研究採用缺乏莢膜多醣體表面因子的突變株JF046 (ΔORFVV0364) 與野生株YJ016,分析其細菌生長特性與生物膜形成量;並且以qRT-PCR測量群體感應調控子LuxR與SmcR基因的mRNA表現量,進行分析比較。結果顯示:(一)、在菌落型態上,YJ016為不透明菌落,而JF046為半透明菌落。(二)、細菌生長曲線的結果顯示,(A600) 無論是在海水環境 (經24小時培養,YJ016為1.795,JF046為1.486;p=0.006),或淡水環境 (經24小時培養,YJ016為1.519,JF046為1.141;p=0.005),莢膜多醣體突變株JF046的生長密度都比野生株YJ016低。(三)、在72小時培養後,莢膜多醣體的突變株JF046的生長會產生細菌聚集體,而野生株YJ016的生長則無。(四)、在生物膜形成的分析試驗中,發現生物膜形成能力(A492/A620),無論是在海水環境 (YJ016為0.91±0.15,JF046為1.50±0.23;p=0.047),或淡水環境 (YJ016為0.50±0.05,JF046為1.26±0.20;p=0.013) ,莢膜多醣體突變株JF046的生物膜形成能力都比野生株YJ016高。(五)、在群體數量感應基因LuxR/SmcR的mRNA表現方面,分析群體數量感應調控子LuxR 與SmcR基因的表現相關性,以ANOVA回歸分析的結果顯示:在野生株YJ016 的r2 =0.885 (y=1.045x+7.798),在突變株JF046的r2 = 0.798 (y=0.928x+25.835)。本研究綜合以上結果證明創傷弧菌莢膜多醣體表面因子ORFVV0364會影響生物膜形成及群體數量感應調控子LuxR/SmcR的表現。

英文摘要

Vibrio vulnificus is a halophilic gram-negative bacterial species. In nature, V. vulnificus is present in marine environment and causes opportunistic infections in humans including septicemia and wound infection. The capsular polysaccharide is one of potential virulence factors and associated with formation of biofilm. To understand the effect of capsular polysaccharide on the formation of biofilm, this study uses the acapsular strain JF046 ( ORFVV0364) with a mutation in capsular polysaccharide surface factor. The characteristics of bacterial growth and formation of biofilm were analyzed between strains. Furthermore, the mRNA expression of quorum-sensing regulators including LuxR and SmcR were determined and compared. First, the colony of YJ016 appeared to be opaque and JF046 was translucent. Second, the results of growth curve indicated that the bacterial density for growth (A600) were lower in JF046 than those in YJ016 both in seawater environment (cultured for 24 hours, 1.795 for YJ016, 1.486 for JF046; p=0.006) and fresh water environment (cultured for 24 hours, 1.519 for YJ016, 1.141 for JF046; p=0.005), respectively. Third, the bacterial aggregates were existed in JF046 and absent in YJ016. Fourth, the ability for formations of biofilm (A492/A620) in acapsular mutant JF046 were higher than those in wild type YJ016 both in seawater environment (0.91 0.15 for YJ016, 1.50 0.23 for JF046; p=0.047) and fresh water (0.50 0.05 for YJ016, 1.26 0.20 for JF046; p=0.013). Fifth, the expressions of quorum-sensing regulators, LuxR and SmcR, were compared between JF046 and YJ016. The results form regression analysis revealed that the correlation between mRNA expressions of LuxR and SmcR were r2=0.885 (y=1.045x+7.798) in YJ016 and r2=0.798 (y=0.928x+25.835) in JF046, respectively. In conclusion, the results demonstrated that the ORFVV0364 for capsular polysaccharide surface factor of Vibrio vulnificus is associated with formation of biofilm and quorum-sensing regulators expression.


指導教授 - 黃小萍

指導教授 - 羅宏仁

召集委員 - 李瑞年

委員 - 王紹鴻


 

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