克雷白氏肺炎桿菌CG43中PecS與PecM在調控第三型纖毛所扮演的角色

Abstract

在克雷白氏肺炎桿菌CG43菌株，若移除座落於第一型纖毛及第三型纖毛基因組間之pecS或pecM基因，可增加第三型纖毛主結構蛋白MrkA之表現，因此本研究進一步探討pecS與pecM在克雷白氏肺炎桿菌之纖毛調控上所扮演之角色。藉由西方墨點法、甘露糖敏感之酵母菌凝集測試及生物膜生成活性測試，顯示pecS或pecM之基因缺損會導致第三型纖毛之表現量上升，但不影響第一型纖毛之表現，且移除pecM對第三型纖毛表現量之影響似乎比移除pecS更顯著。此外，添加尿酸會使MrkA表現量及生物膜生成活性皆明顯上升，但將pecM基因移除後，其生物膜之生成活性卻無顯著上升，暗示具穿膜結構之PecM可能參與尿酸之運送，而尿酸可誘導生物膜之生成。續以凝膠電泳位移分析，證實重組蛋白PecS可結合含有類pecO序列之mrkA、pecS和pecM之啟動子，且在凝膠電泳位移競爭實驗，證實尿酸可作為配體角色，防止PecS與含有pecO序列之DNA結合。在啟動子活性之量測試驗，發現一旦pecS缺失，pecS與pecM啟動子之活性顯著上升，此結果證實PecM和PecS除了會抑制第三型纖毛之表現，PecS亦可在轉錄層面抑制pecS及pecM基因之表現。另亦證實PecM或PecS之基因缺損似乎並不影響細菌抵抗paraquat所造成之氧化壓力，且利用Two hybrid分析，顯示PecS與PecM之間似乎無交互作用。

In Klebsiella pneumoniae CG43, deletion of pecS or pecM, which located in-between the type 1 and type 3 fimbriae gene clusters, had previously been shown to increase the expression of type 3 fimbriae major pilin MrkA. Here the deleting effect of pecS and pecM is further analyzed. The analysis of western blotting hybridization, mannose sensitive yeast agglutination, and biofilm formation activity revealed that either gene deletion increased the expression of type 3 fimbriae but had no apparent influence on type 1 fimbriae expression. Notably, the deleting effect of pecM on type 3 fimbriae was more apparent than that of pecS. Addition of urate was able to increase the MrkA expression and biofilm forming activity, however, deletion of the pecM had lost of the urate induced activity. This implied that the transmembrane domain of PecM may be involved in the transport or sensing uric acid for biofilm formation. Moreover, EMSA analysis indicated that the recombinant PecS was able to bind to the the putative pecO-containing promoters PmrkA, PpecS or PpecM. The following competitive assay suggests that uric acid is a ligand of the recombinant PecS to prevent from the binding to the pecO-containing DNA. Moreover, the promoter activity measurement revealed that the level of PpecS, PpecM, or PmrkA increased by the deletion of pecS. Taken together, these results suggest that PecM and PecS are able to negatively regulate the expression of type 3 fimbriae and PecS could also repress pecS and pecM expression at the transcriptional level. Finally, disc inhibition assay failed to detect either gene deleting effect on the bacterial response to paraquat treatment. The 2-hybrid analysis was also unable to demonstrate the interaction between PecS and PecM.