克雷伯氏肺炎桿菌第六型分泌系統之調控

Abstract

細菌會利用不同的致病因子(virulence factor)來抵禦或逃脫宿主的免疫攻擊，並提升在宿主環境中生存或造成威脅的能力，而細菌分泌蛋白質的過程為一種與宿主互動的重要策略。已知革蘭氏陰性菌演化出六型分泌系統(type I to type VI secretion system, T1SS to T6SS)，而第六型分泌系統是最近期所發現，會透過組裝精密的Hcp-VgrG-PAAR突刺裝置，將作用蛋白質(effector protein)運輸至目標細胞或環境中。據研究指出高於25%的革蘭氏陰性菌帶有構成T6SS核心構造之13個保守基因，而本篇研究對象克雷伯氏肺炎桿菌NTUH-K2044也被發現有T6SS locus-I和locus-III的存在，故想深入探討其T6SS是否具有功能，且會受何種未知機制所調控。 本研究結果顯示，T6SS相關基因(hcp、vgrG1、vgrG2、icmF1和icmF2)廣泛分布於不同來源的克雷伯氏肺炎桿菌臨床菌株，其中以分離自社區型化膿性肝膿瘍(PLA)菌株同時帶有此五個基因之比率為最高（32/42, 76.19%），但隨機挑選的47株hcp+vgrG1+vgrG2+icmF1+icmF2+菌株，包括野生型NTUH-K2044，並未在西方墨點法偵測到Hcp蛋白質的表現，故認為克雷伯氏肺炎桿菌之T6SS在一般實驗室培養條件下可能受細菌內未知機制所壓抑。接續利用即時定量聚合酶鏈鎖反應分析發現H-NS蛋白質會在轉錄層次上負向調控hcp、vgrG1和vgrG2 mRNA表現量，且以西方墨點法在hns基因剔除株中可偵測到Hcp蛋白質的表現。另外，野生型NTUH-K2044在與真核細胞(HL-60, Caco-2 cells)作用後，其hns mRNA表現量雖有顯著降低，但並無提升T6SS相關基因的表現，顯示T6SS在NTUH-K2044與真核細胞作用之下會受到更嚴謹且未知的機制所調控。接著利用凝膠阻滯分析法確認H-NS可直接與T6SS locus-I上之啟動子序列或是基因編碼序列相互結合，藉此抑制T6SS相關基因的表現。最後，在與Caco-2細胞貼附實驗中，發現T6SS相關基因會影響NTUH-K2044對腸道細胞的貼附能力，而其中詳細的分子機制為何仍需更深入的研究。

Bacteria can use different virulence factors to resist or escape host immune response, even to enhance survival ability in host environment. Currently, Gram-negative bacteria have been known to evolve six types of secretion system (T1SS to T6SS), wherein the type six secretion system (T6SS) was discovered recently, which can transport effector proteins to target cells or environment through assembling a delicate Hcp-VgrG-PAAR needle-like structure. Several studies have indicated that up to 25% of Gram-negative bacteria genome includes 13 conserved genes, which encoded the core components of T6SS. In fact, two contiguous putative T6SS loci (locus-I, locus-III) have been found in the Klebsiella pneumoniae NTUH-K2044 genome in a previous study. Therefore, the aim of this study is to investigate whether the NTUH-K2044 have functional T6SS or not, and to identify the unknown regulation mechanisms of its T6SS. Our results showed that five of the T6SS genes (hcp, vgrG1, vgrG2, icmF1, icmF2) that were highly related to the assembly and function of T6SS were widespread in various clinical isolates of K. pneumoniae, and strains isolated from community-acquired pyogenic liver abscess (PLA) have a higher prevalence (32/42, 76.19%). The Hcp protein was detected neither in randomly selected 47 hcp+vgrG1+ vgrG2+icmF1+icmF2+ strains nor in the wild-type NTUH-K2044. Thus, we assumed that T6SS was repressed via unknown mechanisms in general in vitro culture conditions. Next, we found that transcription levels of hcp, vgrG1 and vgrG2 were suppressed by H-NS via real-time PCR analysis. The Hcp protein expression was significantly increased in the hns deletion mutant strain. The hns transcription level of the NTUH-K2044 was significantly decreased after interacting with both HL-60 and Caco-2 cells, however, the expression of T6SS-related genes did not increased, revealing that a more stringent and unknown mechanisms behind when interacting with eukaryotic cells. In addition, the gel retardation assay confirmed that H-NS could bind to the promoter sequences or coding region of T6SS locus-I, indicating the suppression of transcription of T6SS-related genes is through direct binding. At last, we found that T6SS-related genes were involved in the NTUH-K2044 adhesion to Caco-2 intestinal cells from adherence assays, while the detailed molecular mechanisms still need further study.