||Role of a small RNA, VrsA, in regulation of Vibrio vulnificus virulence
||Department of Microbiology & Immunology
創傷弧菌是一種革蘭式陰性致病菌，會透過傷口感染或吃進帶菌的食物造成壞死性筋膜炎和猛爆性敗血症。我們實驗室已經發現了一個廣泛調控子─ 白胺酸反應調控蛋白質（Lrp）參與調控一株臨床菌株YJ016的趨化性、細胞毒性和對小鼠的毒力。為了瞭解Lrp如何調控創傷弧菌對小鼠之毒力，我們比較了野生株YJ016和Δlrp突變株培養在80%小鼠血清中2小時之全轉錄體，找到了在Δlrp突變株中呈現與野生株不同轉錄水平的Lrp目標基因。為了進一步探討是否有小RNA（sRNA）參與在Lrp的毒力調控機制，在本研究中，我們從RNA-seq的原始數據中尋找在Δlrp突變株表現出不同轉錄水平的sRNA，因此找到了一個在Δlrp突變株中轉錄水平上升的sRNA，將之命名為VrsA。VrsA是從VV2389和VV2390兩基因之間的區域轉錄出來，長度大約400 nt，並且和這兩個基因具同一轉錄方向。我們把一株在Lrp的DNA結合區域有一個點突變並且和Δlrp突變株有相似表現型的菌株YH06中的vrsA刪除後，發現此YH06ΔvrsA突變株在軟瓊脂上的移動性、細胞毒性和小鼠血清中的生長均改變了。在回補了VrsA之後，這株細菌在軟瓊脂上的移動性和細胞毒性上又都恢復到和YH06相當，但其在小鼠血清中的生長則較YH06為佳。另一方面，在野生株YJ016中大量表達VrsA並不會造成表現型的改變，只除了其在小鼠血清中的生長較野生株為佳，而和YH06ΔvrsA回補株相當。這些結果表示VrsA可能參與在Lrp的毒力調控中。不過，VrsA並不是藉由影響Lrp的表現量來調控這些性狀，因為在VrsA缺乏和過度表達的菌株裡，Lrp的量和表現型的變化並沒有相關性。我接著發現，一些和趨化性、細胞毒性、攝鐵能力相關的基因在YH06ΔvrsA突變株中的表現量都會上升，而在回補株中表現量又會下降到和YH06一樣，與上述此兩菌株表現型變化的情形一致。最後，通過生物資訊軟件TargetRNA2的分析，找到了一些VrsA的可能目標基因，其中有三個與毒力相關的基因已經確認會受到VrsA的調控。
Vibrio vulnificus, a gram-negative bacterial pathogen, can cause necrotizing fasciitis and fulminant septicemia in humans via wound or food-borne infection. Our laboratory has shown that the leucine-responsive transcriptional regulator (Lrp), a global regulator, is involved in the regulation of chemotaxis, cytotoxicity and virulence in the mouse in a clinical strain YJ016. To understand how Lrp regulates the virulence of V. vulnificus, the target genes of Lrp, which showed altered transcriptional levels in the Δlrp mutant, were identified by comparing the transcriptome of this mutant incubated in 80% mouse serum for 2 hours with that of its parent strain YJ016. To further explore whether small RNAs (sRNAs) may be involved in the regulation of virulence by Lrp, sRNAs with altered expression levels in the Δlrp mutant were searched by analyzing the RNA-seq raw data in this study. A sRNA, designated VrsA, upregulated in the Δlrp mutant was thus identified. VrsA was transcribed from the intergenic region between VV2389 and VV2390 in the same direction as these two flanking genes, and was estimated to be about 400 nt in length. Deletion of vrsA from mutant YH06, which contains a missense mutation in the DNA-binding domain of Lrp and showed similar phenotype as the Δlrp mutant, resulted in phenotypic change in migration on soft agar, cytotoxicity and growth in mouse serum. The complemented strain of this mutant resembled mutant YH06 in migration on soft agar and cytotoxicity, but grew much better than YH06 in mouse serum. On the other hand, overexpression of VrsA in the wild-type strain did not cause phenotypic change except for growth in mouse serum, which was similar to the complemented strain of the YH06vrsA mutant. These results suggest that VrsA may be involved in the regulation of virulence by Lrp. Nevertheless, VrsA does not regulate via affecting the Lrp level, because the Lrp levels in the VrsA-deficient and -overexpressing strains did not correlate with the phenotype. Consistently, some of the genes involved in chemotaxis, cytotoxicity and iron-acquisition were up-regulated in the YH06vrsA mutant but restored to mutant YH06 levels in the complemented strain. Finally, a number of candidate target genes of VrsA were identified by a bioinformatic tool, TargetRNA2. Among them, three virulence-related genes were confirmed to be regulated by VrsA.
List of tables VII
List of figures VIII
Materials and Methods 6
1. Bacterial strains, plasmids, primers and cell lines 6
1.1 Bacterial strains and plasmids 6
1.2 Primers 6
1.3 Cell lines 6
2. Nucleic Acid extraction and manipulation 6
2.1 Genomic DNA extraction 6
2.2 Plasmid DNA extraction 7
2.3 Polymerase chain reaction (PCR) 7
2.4 Splicing by overlap extension 7
2.5 DNA clean up and gel extraction 8
2.6 Restriction enzyme digestion 8
2.7 Dephosphorylation of 5’-ends of DNA 8
2.8 DNA ligation 9
2.9 Competent cell preparation 9
2.10 Transformation 9
2.11 Conjugation 9
2.12 RNA isolation and reverse-transcription PCR 10
2.13 Real-time RT-PCR 10
3. Isolation of mutants 11
3.1 Isolation of YJ016Δlrp mutant 11
3.2 Isolation of the vrsA deletion mutants 11
3.3 Isolation of the VrsA-complemented strains 12
3.4 Isolation of the VrsA overexpressing strain 12
3.5 Isolation of strains expressing transcripts with a His6-tag 12
4. Phenotype Analysis 13
4.1 Migration on soft agar 13
4.2 Cytotoxicity assay 13
4.3 Bacterial growth curves 13
5. Protein analysis 13
5.1 Preparation of bacterial total proteins 13
5.2 Estimation of protein concentration 14
5.3 Western blot analysis 14
6. Animal experiments 14
6.1 Mice 14
6.2 Growth curve of bacteria incubated in mouse serum 15
6.3 Survival rate of infected mice 15
7. Statistical analyses 15
1. Identification of the small RNA, VrsA, that showed altered transcriptional level in the lrp mutant 16
2. Direction of vrsA transcription 16
3. Length of VrsA 17
4. The hypothetical ORFs in vrsA are not translated 17
5. Phenotype of the lrp mutant with a deletion in vrsA 17
5.1 Isolation of the YJ016lrp*ΔvrsA mutant and complemented strain 18
5.2 Involvement of VrsA in reduced migration of YH06 (YJ016lrp*) on soft agar 18
5.3 Involvement of VrsA in cytotoxicity defect of YH06 (YJ016lrp*) 19
5.4 Growth of YJ016lrp*ΔvrsA mutant and its complemented strain in vitro and ex vivo 19
6. Phenotype of the wild-type strain overexpressing VrsA. 19
6.1 Isolation of YJ016(pvrsA) that overexpressed VrsA 19
6.2 Phenotype of YJ016(pvrsA) 20
7. Effect of VrsA on the expression of Lrp 20
7.1 The lrp mRNA levels in the VrsA-deficient and -overexpressing strains 20
7.2 The Lrp protein levels in the VrsA-deficient and -overexpressing strains 20
8. The mRNA levels of the up- and down-stream genes of vrsA in the YH06ΔvrsA mutant 20
9. Chemotaxis, cytotoxin and iron acquisition-associated genes regulated by VrsA 21
9.1 The mRNA levels of chemotaxis-associated genes in YJ016lrp*ΔvrsA 21
9.2 The mRNA levels of cytotoxin genes in YJ016lrp*ΔvrsA 21
9.3 The mRNA levels of iron acquisition-associated genes in YJ016lrp*ΔvrsA 21
10. Identification of putative target genes of VrsA 22
Tables and Figures 36
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