尿道致病奇異變形桿菌N2第六型分泌系統之研究

Abstract

Proteus mirabilis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。為了存活，細菌發展出許多調控機制，來適應環境中各種變化。第六型分泌系統（T6SS）是細菌間用來彼此競爭的武器之一，除了在同種及不同種菌之間的競爭外，T6SS亦會作用在真核細胞上。先前文獻指出，T6SS會受到與環境相關的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。但有關T6SS在P. mirabilis當中的調控情形及作用尚待釐清，因此本研究利用P. mirabilis的regulators及sigma factors突變株來探討其對T6SS的影響。 從transcriptome dRNA-seq的結果發現，Crp、Hfq、RpoN、RpoE 及CpxR會影響P. mirabilis N2的T6SS相關基因的表現。透過swarming assay發現野生株會跟上述突變株形成boundary（Dienes line），辨認為不同菌株。利用生長優勢實驗發現野生株皆較具生長優勢。接著透過短時間殺菌試驗觀察到突變株都會被野生株所殺。進一步利用real-time RT-PCR及promoter reporter assay證明Crp及Hfq會正向調控T6SS結構基因。而T6SS會利用hcp-vgrG effector operons執行其功能，在這方面，Crp、Hfq、RpoN、RpoE 及CpxR都會正向調控各套hcp-vgrG effector operons。進一步探討Crp對T6SS的調控，透過crp互補株再次驗證Crp確實會正向調控T6SS結構基因及各套effector operons，也利用DNase I footprinting證明Crp會直接結合在T6SS結構基因及各套effector operons的啟動子區域，並透過殺菌試驗發現crp過度表現株具有較強的殺菌能力。在toxin的部分，我們發現Crp也會影響T6SS中三個可能的toxin gene的表現，其中2040及1472過度表現株使其對P. mirabilis及E. coli均展現了較強的殺菌能力。 綜合上述結果我們認為Crp、Hfq、RpoN、RpoE 及CpxR皆會調控P. mirabilis N2 T6SS的表現，其中以Crp的影響最為顯著。此外，過度表現Crp及T6SS相關toxins會使其展現較強的殺菌能力。

Proteus mirabilis is a frequent pathogens causing urinary tract infection, mainly in patients with the long-term use of urinary catheters. Bacteria have developed diverse regulatory mechanisms for adaptation to the changing environments. Type VI secretion systm (T6SS) is one of the weapons for different bacteria to compete with each other and gain predominance in their niches. It is a protein secretion system which requires cell-cell contact, between bacteria or a bacterium and a eukaryotic cell. Studies have shown that T6SS is regulated by environment-related regulators, but the regulation of T6SS in P. mirabilis remains uncleared. Our transcriptome analysis showed T6SS of P. mirabilis N2 including T6SS main structure gene and four hcp-vgrG effector operons is likely regulated by Crp, Hfq, CpxR, RpoN and RpoE. We observed dienes lines formation between wild-type and crp, hfq, cpxR, rpoN and rpoE mutant strains. The wild-type has growth predomince over all mutant strains and all mutant strains are killed by the wild-type. It means all above regulators and sigma factors affect killing ability of P. mirabilis. We found the P. mirabilis genome contains one T6SS main structure (17 genes) operon and four T6SS hcp-vgrG effector operons by sequence specific reverse transcription PCR. Realtime PCR and promoter reporter assay demonstrated expression of T6SS main structure and/or four hcp-vgrG effector were up-regulated by Crp, Hfq, CpxR, RpoN and RpoE. We further found that Crp directly binds to the promoter regions of T6SS structure and four effector operons by DNase I footprinting analysis. In addition, crp-overexpression strain showed better killing ability against the wild-type. We used bioinformatic tools and found 3 putative toxin genes in T6SS of P. mirabilis N2. Toxin-overexpression strain also exhibited better killing ability than the wild-type and E. coli. Altogether, our study suggests that Crp, Hfq, CpxR, RpoN and RpoE positively regulated T6SS of P. mirabilis N2 and affected its killing ability.