克雷白氏肺炎桿菌CG43中第六型分泌系統-I組成蛋白質Hcp、ClpV和VgrG之功能性探討

Abstract

第六型分泌系統(Type VI secretion system；T6SS)由Hcp和VgrG兩種主要結構蛋白質所構成，並以ClpV蛋白質之ATP水解酶活性為系統運作之主要能量來源，且已被證實與細菌之致病能力密切相關。先前之研究報導指出，在以小鼠為動物模型之研究中，將克雷白氏肺炎桿菌之T6SS基因進行插入突變，會使其感染小鼠之能力減弱。其他菌種之基因體中通常含有多組T6SS基因群集，在克雷白氏肺炎桿菌具有2-3套T6SS基因組。本論文旨在探討克雷白氏肺炎桿菌CG43之T6SS-I組成蛋白Hcp、ClpV和VgrG的功能性。首先，以同源置換原理建構了CG43S3 hcp、CG43S3 clpV、CG43S3 vgrG和VgrG C端基因片段缺失(CG43S3 vgrG –C)突變株，接著分析這些突變菌株與其親本株CG43S3之表現型後發現：hcp和vgrG之生長曲線和菌落型態無明顯變化，clpV之生長則較為遲緩，且其莢膜多醣生成量降低約35%；hcp和vgrG於玻璃材質介面上生物膜形成能力明顯上升，而clpV和vgrG於PVC界面上形成的生物膜量增加約1.7倍；hcp、clpV、vgrG和vgrG -C在酸性環境壓力下存活率分別下降70%、21%、90%和68%。進一步以LacZ報導系統分析T6SS-I基因組中經預測的啟動子P1、P2和P3的活性，結果顯示P1活性會被弱酸誘導，此暗示T6SS-I由P1啟動子調控其表現。為了偵測T6SS-I的表現，先以PCR增幅其主要結構蛋白基因hcp並選殖於表現載體pET30b，接著將此重組質體pET30b-hcp於大腸桿菌Noveblue(DE3)中大量表現後以鎳離子樹脂來純化此His-標記之Hcp蛋白質，最後，以此純化後的蛋白多次注射兔子後獲得Hcp之多株抗體。然而，以西方墨點法分析結果顯示此多株抗體專一性和靈敏度都不足。未來可以定量PCR量測hcp、clpV和vgrG於酸環境中，或於抗酸反應相關的調控基因缺失之菌株：如rcsB或fur中的轉錄表現量，藉以了解T6SS-I的表現調控。

The type six secretion system(T6SS), which composed of two major structural proteins Hcp and VgrG, and the energy generating ATPase ClpV, has been reported as an important pathogenicity factor. Using a mouse model, an attenuated Klebsiella pneumoniae mutant with a transposon insertion in the putative T6SS encoding gene has recently been reported. As reported for other bacterial genomes which often contain multiple T6SS-encoding gene clusters, the sequenced K. pneumoniae genomes also harbored two to three T6SS gene clusters. Here we study functional roles of Hcp, ClpV and VgrG of the T6SS-I of K. pneumoniae CG43. Firstly, we generate K. pneumoniae CG43S3 hcp, CG43S3 clpV, CG43S3 vgrG and CG43S3 vgrG -C (carrying an incomplete vgrG gene without the DNA coding for the C-terminal variation region) mutant strains. The deletion effect analysis showed that CG43S3 hcp and CG43S3 vgrG exerted similar phenotype on growth curve and colony appearance as that of the parental strain CG43S3. On the other hand, the deletion of clpV retarded the bacterial growth and had 35% decrease of the capsular polysaccharide production. The mutants hcp and vgrG exhibited elevated biofilm formation ability on glass tubes, while the deletion of clpV and vgrG increased approximately 1.7-fold of the biofilm formation activity on PVC 96-well compared to CG43S3. Under acid stress treatment, hcp, clpV, vgrG and vgrG-C respectively showed 70%, 21%, 90% and 68% decrease of the survival rate. We have also measured the activity of the putative promoters namely P1, P2 and P3 using LacZ as the reporter. The P1 activity is inducible by weak acid which suggesting that the expression of T6SS-I is P1-dependent. Finally, we have isolated the major component encoding gene hcp by PCR and the amplicon cloned to the expression vector, pET30b. The recombinant plasmid pET30b-hcp was introduced for the protein overexpression in Escherichia coli Novablue(DE3), and then the recombinant His-tagged proteins purified using nickel resin column. Aliquot of the purified proteins was used to immunize rabbits to raise anti-Hcp antibody. The subsequent Western blot analysis using the anti-Hcp antibody revealed that the antibody has low specificity and sensitivity. In order to study the expression of T6SS-I, quantitative PCR could be used to measure the expression level of hcp, clpV and vgrG in CG43S3 upon treatment of acid or in acid stress response related regulatory gene deletion mutants such as rcsB or fur.