cAMP受體蛋白質與TssKFis蛋白質調控尿道致病性奇異變形桿菌 第六型分泌系統之研究

Abstract

Proteus mirabilis為革蘭氏陰性的兼性厭氧菌，是造成泌尿道感染的病原菌之一，主要在長期使用尿導管的病人中造成伺機性感染。細菌發展出許多調控機制，適應環境中各種變化，以利自身生存。第六型分泌系統 (Type VI Secretion System, T6SS) 是細菌間用來彼此競爭的武器之一，除了在同種及不同菌種之間競爭外，T6SS亦會作用在真核細胞上。參考文獻指出，T6SS會受到環境相關或自身操縱組的regulators所調控，且T6SS所分泌的effector proteins (toxins) 的種類及功能有很多，原核及真核細胞都可能為其作用標的。 本實驗室先前發現P. mirabilis中Crp 會正向調控T6SS結構基因及各套effector operons的表現，並在殺菌試驗中觀察到crp突變株會受野生株所殺，而crp過度表現株則會殺野生珠。VipA及VipB為包覆在Hcp外圍的管狀外鞘，且此外鞘具有收縮性，藉由VipA/B的伸縮可將上述穿刺裝置打出。因此本研究建立vipAB突變株來探討Crp參與殺菌試驗之過程，觀察到野生株與crp過度表現株皆須需透過T6SS攻擊菌株。 vipAB突變株中gentamicin resistant cassette含terminator，因此利用real-time PCR調查出vipAB突變會影響下游T6SS結構基因表現，且有三組hcp-vgrG effector operons基因表現會大幅受到抑制。接著透過Protein BLAST，發現基因編號2057編碼Fis family transcriptional regulator (TssKFis)。首先藉由tssKfis過度表現，探討TssKFis在P. mirabilis對於T6SS的調控。在reporter assay中，tssKfis過度表現會使得野生株中三組hcp-vgrG effector operons的promoter活性上升；T6SS結構操縱子則下降。再者，利用tssKfis突變株觀察到P. mirabilis N2中三組hcp-vgrG effector operons確實會受到TssKFis調控。 綜合上述結果，藉由vipAB突變證實Crp會透過調控P. mirabilis N2中T6SS的表現，來參與殺菌過程。此外，我們推論T6SS結構操縱子中的TssKFis會調控T6SS的表現。

Proteus mirabilis with the swarming characteristic often causes urinary tract infections occurring mainly in patients with the long-term implantation of urinary catheters. Bacteria have developed many regulatory mechanisms to adapt to environmental changes and pressures for survival. The type VI secretion system (T6SS), a widespread multi-protein machine in Gram-negative bacteria, delivers effectors to compete with other bacteria or infect eukaryotic hosts. Based on the analysis of P. mirabilis N2 transcriptome, T6SS is regulated by a global regulator, cAMP receptor protein (Crp). Therefore, we investigate the role of Crp in T6SS expression and associated functions. Our previous studies delineated that Crp positively regulates T6SS structure and hcp-vgrG effector operons in P. mirabilis. Furthermore, crp would be attacked by wild-type; wild-type got hurt by crp overexpression strain in killing assay. VipA and VipB are contractile sheath proteins surrounding Hcp tube and contraction of the VipA/B pushes the Hcp-VgrG needle out of the cell. In this study, we established vipAB strain to investigate Crp-mediated killing process and found that T6SS is necessary for wild-type and crp overexpression strain to compete with prey strains。 Due to the presence of a transcriptional terminator downstream the gentamicin resistant cassette (gmc) in vipAB, we confirmed the impairment of gene expression downstream the gmc by the real-time PCR. Among them, we found that gene number 2057 coding Fis family transcriptional regulator (TssKFis) in P. mirabilis by the Protein BLAST. In the beginning, we utilized tssKfis overexpression to investigate the regulation of TssKFis to T6SS in P. mirabilis. tssKfis overexpression increased promoter activity of three hcp-vgrG effector operons and decreased the T6SS structure operon by reporter assay. We found vipAB defect also dramatically decreased mRNA level of hcp-vgrG effector genes. Recently, we constructed tssKfis to prove that three of four hcp-vgrG effector operons be be regulated by TssKFis in P. mirabilis N2. In conclusion, we demonstrate that T6SS is critical for Crp mediated killing process in P. mirabilis N2. Moreover, TssKFis in T6SS structure operon can regulate T6SS expression.