Kva15在克雷白氏肺炎桿菌CG43之 cyclic di-GMP調控路徑中的角色

Abstract

克雷白氏肺炎桿菌CG43是從患肝膿瘍的糖尿病病人身上分離的臨床菌株，在先前轉錄體分析實驗發現提高CG43的c-di-GMP濃度，其轉錄因子kva15的表現量會上升，本論文旨在釐清Kva15是否及如何參與c-di-GMP之調控網路，因為剔除kva15基因對CG43S3胞外多醣生成沒有明顯影響，轉而在有較高c-di-GMP濃度的CG43S3∆yjcC移除kva15，結果使其胞外多醣生成明顯降低，而增加Kva15表現會提升胞外多醣生成的現象在剔除負責分泌poly-N-acetyl glucosamine（PNAG）的pgaC基因後不復見，進一步以免疫分析試驗確認在CG43S3或CG43S3∆yjcC中移除kva15均可降低PNAG的表現，顯示受Kva15調控表現的胞外多醣是PNAG。剔除kva15基因對CG43S3只稍微降低第三型線毛單位蛋白MrkA的表現，而在有較高c-di-GMP濃度的CG43S3∆yjcC移除kva15會使其MrkA表現明顯降低，暗示Kva15會正向調控MrkA表現。但是，啟動子活性分析結果顯示kva15基因缺失並不影響pgaABCD和mrkA的表現，提高c-di-GMP濃度之下也看不出kva15基因缺失對二者啟動子活性的影響，此結果暗示Kva15對PNAG及第三型線毛表現的影響可能不是在轉錄階段；接著，將kva15表現質體植入大腸桿菌BW25113後觀察對其泳動能力，結果顯示kva15表現會降低泳動能力，暗示Kva15正向調控c-di-GMP濃度進而影響細菌表現型；此外，以螢光亮度反映c-di-GMP濃度的實驗也支持Kva15正向影響c-di-GMP的可能性；透過啟動子活性分析發現kva15基因缺失會增加yjcC的表現活性，這意味著Kva15可能透過負向調控yjcC表現來提升c-di-GMP濃度。最後，由於在kva15的啟動子序列上可預測到Fur及HNS的結合位點，以啟動子活性分析發現kva15與pgaA的活性在fur及hns基因缺失下皆會增加，然而fur的影響不受鐵離子移除而變化；此外，在CG43S3 ∆fur∆kva15剔除ryhB後會使胞外多醣與MrkA表現降低，此暗示ryhB參與Fur-Kva15的調控途徑，其可能性還待進一步研究。

Klebsiella pneumoniae CG43 is a clinical isolate from a diabetic patient with liver abscess. Previous transcriptomic analysis in K. pneumoniae CG43 revealed that the expression of the transcription factor encoding gene kva15 is enhanced by the increase of the c-di-GMP levels. Here, how Kva15 is involved in the c-di-GMP regulatory pathway is studied. Deletion of kva15 had no apparent effect on the production of extracellular polysaccharides (EPS). Nevertheless, deleting kva15 from CG43S3∆yjcC which has higher c-di-GMP levels than CG43S3, apparently decreased the production of EPS. The increasing EPS production was no longer observed when the poly-N-acetyl glucosamine (PNAG) encoding gene pgaC was deleted. Deletion of kva15 in CG43S3 and CG43S3∆yjcC decreased the production of PNAG using dot blot assay further suggesting the Kva15 dependent production of EPS is PNAG. Deletion of kva15 slightly decreased the expression of the type 3 fimbriae major pilin MrkA. Moreover, deleting kva15 from CG43S3∆yjcC also decreased the production of EPS and MrkA. However, deletion of kva15 had no apparent effect on the promoter activity of pgaABCD and mrkA even in the background of high c-di-GMP level. These results suggest that the influence of Kva15 may not be at the transcription level. Introducing the kva15 expression plasmid into E. coli BW25113 was found to reduce the swimming activity implying that Kva15 influencing the bacterial phenotypes via positively modulating the c-d-GMP levels. Moreover, deletion of kva15 had positive effect on the promoter activity of yjcC indicating a negative role of Kva15 on the yjcC expression thereafter increasing the c-di-GMP levels. Besides, the analysis of the fluorescence reflecting the c-di-GMP levels also supports that Kva15 plays a positive role in regulating the c-di-GMP levels. Since a conserved Fur and HNS binding elements could be identified in the putative promoter, kva15 expression in CG43S3Z01∆fur and CG43S3Z01∆hns was then determined. The expression of kva15 and pgaA were both increased by removing fur and hns gene, however, iron depletion had no effect on the Fur-mediated expression. Moreover, the EPS and MrkA production of CG43S3∆fur∆kva15 was apparently decreased by removing the small RNA encoding gene ryhB suggesting RyhB is involved in the Fur-Kva15 regulatory pathway. Nevertheless, the possibility remains to be studied.