T6SS-I 與調節壓力相關之轉錄調控因子蛋白之關係 目前相關 研究證實不同菌種之 T6SS 可受不同環境壓力誘導表

現，而不同轉錄調控蛋白對 T6SS 的調控機制亦迥異。如圖十三 B 之 左圖顯示，西方墨點法分析rpoS、soxRS、pecS 和pecSpecM 的 全蛋白圖譜與 CG43S3 並無明顯差異；有趣的是fur 的全蛋白圖譜與

24

CG43S3 或其他菌株明顯不同，缺少了 18 kDa 蛋白，並於約 30 kDa 位置出現一未知蛋白；而圖十三 B 右圖結果確認回補 fur 基因後，出 現 18 kDa 蛋白，暗示此抗體會非專一地辨認 Fur 蛋白。為了瞭解 T6SS-I 是否與外膜蛋白或第三型線毛的表現相關，如圖十三 C 的西 方墨點分析結果顯示，Hcp 抗體可以非專一性地分辨 OmpA 外膜蛋 白。如圖十四所示，為了降低非專一性的干擾，將抗體稀釋 30000 倍 後的西方墨點法分析結果顯示，雖然非專一性的蛋白帶明顯減少，此 抗體卻無法偵測出於 CG43S3 中大量表現約 34 kDa 的重組 Hcp 融合 蛋白，顯示此抗體的專一性和靈敏度太低。

25 T6SS-III 的組成不完整，缺少 hcp 基因，顯示可能已喪失功能性。CG43 之 T6SS-I 和 NTUH K-2044、MGH78578 依樣具有分別與 T6SS 的結 構形成和傳遞作用之蛋白生成相關的 vgrG 和 icmF gene cluster[65]。

VgrG 之功能域預測分析結果則在其 C 端發現一功能未知之區域，此 VgrG-I 為一 evolved VgrG(圖二)。

clpV 基因缺失使克雷白氏肺炎桿菌 CG43 在對數增殖期的晚期、

生長減衰期和定常期的早期生長較遲緩(圖六 A)，可能因為缺少了 ClpV 的 ATP 水解酶活性，造成生長時能量供給不足，造成菌數增長 較為緩慢、菌落大小不一的現象(圖六 B)，而能量不足亦可能是造成

26 為 T6SS 正常運作所必須，亦為 Y. pseudotuberculosis 對抗酸性環境壓 力，於酸性環境中之存活所必須[60]。克雷白氏肺炎桿菌 CG43 缺失

27

一性不足，無法明確辨識 Hcp 蛋白質；大量表現 hcp 或 fur 之菌株也 無法確認 Hcp 的表現。未來可以再純化(re-purification)方式改善抗體 專一性的問題，有鑑於抗體之專一性和敏感度不足，未來可以 q-PCR 方式探討 T6SS-I 之調控途徑，明確反映 hcp、clpV 和 vgrG 之表現量，

進而描繪出 T6SS-I 之調控途徑。

28

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34

RecA1 supE44 endA1 hsdR17 gyrA96

rolA1 thi (lac-proAB) Lab stock

S17-1λpir

Tp^r Sm^r recA, thi, pro, hsdR^－M^＋ [PR4-2-Tc::Mu:Km^r Tn7](pir)

[77]

CG43yfdX yfdX deletion mutant in CG43, Sm^r Lab stock CG43hcp hcp deletion mutant in CG43, Sm^r This Study CG43clpV clpV deletion mutant in CG43, Sm^r This Study CG43vgrG vgrG deletion mutant in CG43, Sm^r This Study CG43vgrG C-ter rcsD Hk domain deletion mutant in

CG43, Sm^r

This Study CG43rcsB rcsB deletion mutant in CG43, Sm^r Lab stock CG43mrkA mrkA deletion mutant in CG43, Sm^r Lab stock

35

CG43ompA ompA deletion mutant in CG43, Sm^r Lab stock CG43ompC ompC deletion mutant in CG43, Sm^r Lab stock CG43ompF ompF deletion mutant in CG43, Sm^r Lab stock CG43ompN ompN deletion mutant in CG43, Sm^r Lab stock CG43yjcC yjcC deletion mutant in CG43, Sm^r Lab stock CG43mrkH mrkH deletion mutant in CG43, Sm^r Lab stock CG43mrkI mrkI deletion mutant in CG43, Sm^r Lab stock CG43mrkJ mrkJ deletion mutant in CG43, Sm^r Lab stock CG43fur fur deletion mutant in CG43, Sm^r Lab stock CG43rpoS rpoS deletion mutant in CG43, Sm^r Lab stock CG43soxRS soxRS deletion mutant in CG43, Sm^r Lab stock CG43pecS pecS deletion mutant in CG43, Sm^r Lab stock

CG43pecSpecM pecS pecM mutant in CG43, Sm^r Lab stock

CG43lacZhcp lacZ hcp mutant in CG43, Sm^r This study

CG43lacZclpV lacZ clpV mutant in CG43, Sm^r This study

CG43lacZvgrG lacZ vgrG mutant in CG43, Sm^r This study

36

表二：本研究所使用的質體

質體 相關特性

來源或 參考文獻

yT&A PCR cloning vector，Ap^r

Yeastern Biotech Co.

pET30b

Km^r, His-tagged protein expression vector

Novagen Co.

pBAD202

Km^r, His-tagged protein expression vector

Invitrogen Co.

pKAS46 Ap^r, Km^r, suicide vector, rpsL Lab stock placZ15 Cm^r, promoter selection vector, lacZ⁺ Lab stock

pET30b-Hcp

Km^r, His-tagged protein expression vector, 492-bp fragment encoding full-length Hcp cloned into pET30b

This study

pBAD202-Hcp

Km^r, His-tagged protein expression vector, 492-bp fragment encoding full-length Hcp cloned into pBAD202

Y. C. Lai, CSMU

37

(A)

(B)

圖一：克雷白氏肺炎桿菌之 T6SS 基因組成示意圖

38

圖二：Hcp、ClpV 與 VgrG 蛋白質功能區域分析

以線上軟體 Pfam（http://pfam.sanger.ac.uk/）分析 Hcp、ClpV 和 VgrG 之胺基酸序列並對其功能域進行預測。

39

(A)

(B)

圖三：建構 hcp基因缺損突變株

（A）hcp 基因示意圖 hcpCF/hcpCR 為設計來確認 hcp 突變株之引 子；虛線部分表示基因缺損位置。（B）利用 PCR 原理確認 hcp 突 變株。M：DNA 分子大小標記物，WT：野生型菌株，P：帶有突變 片段的 pKAS46 載體，Mu：hcp 突變株。基因缺損的詳細流程於實 驗材料與方法中。

P Mu M WT

500 750 1000 1500 2000

1026 564 bp

40

(A)

(B)

圖四：建構clpV 基因缺損突變株

（A）clpV 基因示意圖 clpVAF/clpVBR 為設計來確認 clpV 突變株之 引子；虛線部分表示基因缺損位置。（B）利用 PCR 原理確認 clpV 突變株。M：DNA 分子大小標記物，WT：野生型菌株，P：帶有突 變片段的 pKAS46 載體，Mu：clpV 突變株。基因缺損的詳細流程於 實驗材料與方法中。

41

(A)

(B)

(C)

42

(D)

圖五：建構vgrG 與vgrG - C 端變異區基因缺損突變株

（A）vgrG 基因示意圖 vgrGCF/vgrGCR 為設計來確認 vgrG 突變株 之引子；虛線部分表示基因缺損位置。（B）利用 PCR 原理確認 vgrG

（A）vgrG 基因示意圖 vgrGCF/vgrGCR 為設計來確認 vgrG 突變株 之引子；虛線部分表示基因缺損位置。（B）利用 PCR 原理確認 vgrG

在文檔中 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 (頁 34-68)