創傷弧菌屬於革蘭氏陰性病原菌，可藉由傷口或食入未煮熟之海鮮而感染人類，會在帶有慢性肝病等潛在性疾病的病人造成嚴重的敗血症和壞死性筋膜炎。我們實驗室先前發現，創傷弧菌中的一個廣泛調控子Lrp能調控此菌對小鼠之毒力，且在Δlrp突變株中發現許多參與在代謝作用、細菌趨化功能及攝取鐵相關之基因的mRNA表現量均顯著下降。為了解Lrp調控細菌毒力的機制，我嘗試找出被Lrp直接調控的目標基因。首先，我分別製備了野生型Lrp及在DNA結合部位發生點突變的Lrp*突變蛋白質之多株抗體，且純化了Lrp-his6重組蛋白質以用在之後的電泳遷移實驗(EMSA)。接著，由EMSA及qRT-PCR的實驗結果顯示，創傷弧菌YJ016菌株中Lrp能直接與tonB3 (VV0250-VV0244)及cadBA (VV2382-VV2383)操縱組之啟動子結合，並活化它們的基因表現，此與先前在其它創傷弧菌菌株中所發現的情形一致。為了找出Lrp在此菌中能直接結合之基因啟動子，我進行了Genome footprinting (GeF)-seq實驗，並使用實驗室之前比較野生株及Δlrp突變株之轉錄體所得的數據，以及創傷弧菌基因體中Lrp所結合之啟動子的預測結果，進一步比對並找出重疊的基因。我由這些基因中選出40個與毒力相關或轉錄調控子之基因，並藉由EMSA實驗確定其中除lrp之外，另有7個包含參與細菌趨化性、攝鐵能力、莢膜合成、胺基酸代謝及轉錄調控作用之基因的啟動子能被Lrp專一性結合，且此7個基因於小鼠血清中培養時，其mRNA表現量在Δlrp突變株中皆較野生株明顯下降，推測可能因此造成Δlrp突變株對小鼠之毒力降低。此外，藉由線上生物資訊軟體之分析，我們在上述10個能被Lrp結合的啟動子中發現一個共有的14 bp長，與大腸桿菌Lrp結合之共同序列相似的DNA序列，推測其為創傷弧菌中Lrp所結合之共同序列。接著，我使用創傷弧菌本身的lacZ作為lrp之啟動子的報導基因，探討Lrp對自身基因之調控，結果發現Lrp的總量隨時間遞增，而lacZ和lrp之mRNA表現量則隨時間遞減，由此推測Lrp對自身基因的調控扮演抑制子的角色。另外，我也發現Lrp與Lrp*對於lrp、VV2382 (cadB)、VV0337及VVA1247的調控能力相差不多，但Lrp*對VV0250 (tonB3操縱組之第一個基因)的調控有些微減弱；透過抗原決定位的預測，也發現Lrp上的點突變可能造成蛋白質結構在N端有些微變化，導致anti-Lrp抗體對Lrp及Lrp*之偵測效果不一樣，因此，我們推測此點突變可能會影響Lrp與DNA之結合，進而影響其調控部分基因之能力。未來可進一步探討本研究中所找到的7個直接受到Lrp調控的基因對細菌之毒力是否扮演重要角色。

Vibrio vulnificus, a gram-negative bacterial pathogen, can infect human via wounds or contaminated seafood and cause necrotizing fasciitis and fulminant septicemia in persons with underlying conditions, such as chronic liver diseases. Our laboratory has previously found that the leucine-responsive regulatory protein (Lrp), a global regulator, in a clinical strain YJ016 is involved in the regulation of virulence in mice. The mRNA levels of many genes associated with metabolism, chemotaxis and iron acquisition were significantly downregulated in the Δlrp mutant. To understand the mechanism of virulence regulation by Lrp, I tried to identify the genes directly regulated by Lrp. First, I prepared the polyclonal antibodies against wild-type Lrp and the Lrp* mutant with point mutation in the DNA-binding domain, and purified the Lrp-his6 recombinant protein to be used in electrophoretic mobility shift assay (EMSA). By EMSA and qRT-PCR analysis, I confirmed that Lrp could bind to the promoters of tonB3 (VV0250-VV0244) and cadBA (VV2382-VV2383) operons to activate their transcription in V. vulnificus YJ016, as had been previously demonstrated in other strains. To identify the promoters directly bound by Lrp in V. vulnificus, I performed genome footprinting by high-throughput sequencing (GeF-seq) and bioinformatic prediction with the consensus sequences of Lrp in E. coli. By comparing between the result of bioinformatic prediction and that of GeF-seq or transcriptome comparison between the wild-type strain and Δlrp mutant conducted previously, I found a number of commonly identified genes. Among them, I selected 40 that were virulence-related or encoding transcriptional regulators. By EMSA, I demonstrated that, other than lrp, 7 genes, including those associated with chemotaxis, iron acquisition, capsule synthesis, amino acid metabolism and transcriptional regulation, were specifically bound by Lrp at the promoter region. The mRNA levels of these 7 genes in the Δlrp mutant were significantly lower than those in the wild-type strain when the bacteria were incubated in mouse serum, and this may partly explain why the virulence of the Δlrp mutant were reduced. The roles of these genes in bacterial virulence are worthy of further investigation. By bioinformatic analysis, we identified a 14 bp DNA sequence, which was similar to the Lrp-binding consensus sequence in E. coli, in all of the ten promoters shown to be bound by Lrp, suggesting that it may be an Lrp-binding consensus sequence in V. vulnificus. I then explored how Lrp regulates the expression of its own gene by using lacZ as a reporter of the lrp promoter. It was shown that the mRNA levels of both lacZ and lrp decreased while the amount of Lrp increased with time, suggesting that Lrp might be a repressor for its own promoter. I also found that the mRNA levels of lrp, VV2382 (cadB), VV0337 and VVA1247 in the presence of Lrp* were similar to those in the presence of Lrp, but, that of VV0250 was lower in the presence of Lrp*. Computational prediction of the epitopes of Lrp and Lrp* suggested that the mutation in Lrp* may slightly change the structure of Lrp at the N-terminus, which could lead to weaker signal of Lrp*, compared to Lrp, upon detection by anti-Lrp. We suspect that the mutation could result in weaker interaction between Lrp* and DNA and loss of regulation ability for some, but not all, genes.

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