克雷白氏肺炎桿菌第一型纖毛和第三型纖毛交互表現的調控

Abstract

細菌纖毛藉由其黏附特異性與特定的細胞結合，為其造成感染的關鍵步驟。已知，第一型或第三型纖毛是克雷白氏肺炎桿菌臨床分離株表現的主要黏附因子。我們在執行94IDP11計劃的實驗發現：在克雷白氏肺炎桿菌株NTUH K2044或 CG43中，第三型纖毛的主要結構蛋白mrkA基因的缺損，明顯增加了第一型纖毛FimA結構蛋白的表現量；而大量表現FimB重組脢，提升第一型纖毛的表現，卻明顯降低了MrkA蛋白的生成；另外，莢膜多醣生成的主要調控蛋白RcsB的基因缺損，降低莢膜多醣的生成，相對的提升第一型纖毛的活性，而MrkA蛋白的表現量卻減少。這些結果顯示第一型纖毛和第三型纖毛的對立表現（counter-expression），也暗示負責著纖毛和莢膜多醣表現的調控系統可能有交互作用。我們在此提出計劃分離鑑定負責此對立表現的調控分子，並探討其對立表現的調控機制。特定的目標如下： 目標一、建構註解為調控基因的特定基因缺損株，探討其對此二纖毛對立表現的影響：這些特定的調控基因包括位於第一型纖毛基因組末端的fimK、位於第三型纖毛基因組前端的phgS、phgM及末端的KP4551、KP4552、KP4554 (參考NTUH K2044的基因體序列註解：http://genome.nhri.org.tw/kp/index.php )。其中，我們將選殖、表現對纖毛活性有明顯缺損影響的調控基因，並探討其對此二纖毛對立表現的調控機制。 目標二、建構跳躍子突變基因庫（transposon-insertion mutant library），篩選可能的調控基因：以mrkA基因缺損株、rcsB基因缺損株為跳躍子接受株時，將以酵母菌凝聚測試（yeast agglutination assay）篩選活性降低的突變株；而以帶有LacZ作為第一、三型纖毛表現的報告系統的菌株為對象時，則以X-Gal培養盤篩選顏色變化的突變株。定序分析這些有明顯變化的突變點後，我們將選殖這些有可能的基因並構築此基因缺損株，再以回補試驗（complementation assay）確認其對第一或第三型纖毛表現的影響，我們也將進一步探討其機制與對莢膜多醣表現的調控交互作用。 目標三、探討外膜逆境反應（Envelope stress responses）的調控在此二纖毛對立表現中可能扮演的角色：mrkA基因缺損株可能因為MrkB、MrkC、MrkD、MrkF蛋白在膜間隙累積而引起外膜逆境，第一型纖毛卻因mrkA基因缺損而表現；另一方面，莢膜多醣的生成只影響第一型纖毛的表現，這些結果暗示外膜環境的調控對第一型、三型纖毛的表現有選擇性的影響。因此，我們將定量分析mrkA基因缺損株中與調控外膜逆境反應相關基因的表現，進一步探討外膜逆境反應在此二纖毛對立表現中可能扮演的角色。

Fimbriae (also called pili) play a key role for bacteria to attach to specific host cell during the establishment of an infection. The expression of type 1 or type 3 fimbriae in the clinical isolates of Klebsiella pneumoniae has been commonly reported. We have found in the previous study (94IDP11) that the deletion of the type 3 fimbrial major subunit encoding gene mrkA of K. pneumoniae NTUH K2044 or CG43 significantly increased the expression of type 1 fimbriae. The overexpression of FimB recombinase was able to switch on the expression of type 1 fimbriae but reduced the MrkA production. Moreover, deletion of the rcsB gene, which encoding a major activator for the biosynthesis of capsule polysaccaharides (CPS), was found to reduce the production of CPS and MrkA, but increased the activity of type 1 fimbriae. These indicated a counter-expression between type 1 and type 3 fimbirae. The possibility of an interacting regulation between fimbriae and CPS biosynthesis was also implicated. It is hence the proposed study is to explore the regulatory mechanisms involved in the control of counter-expression of type 1 and type 3 fimbriae. The specific aims are: 1) To investigate if the deletion of the specific regulatory genes affects the expression: These regulatory genes include fimK, which located at the end of the type 1 fimbrial gene cluster, and phgS and phgM, at the front of the type 3 fimbriae gene cluster, and KP4551, KP4552, and KP4554 (on the basis of the genome annotation of NTUH K2044), at the end of the type 3 fimbriae gene cluster. The regulatory gene with apparent deleting effects on the counter-expression will be cloned and expressed, and the regulatory mechanism elucidated. 2) To generate transposon-insertion mutant libraries to screen for the regulatory genes: The mrkA deletion strain or the rcsB deletion strain will be used as the recipient for the transposon mutagenesis purpose. The mutants reveal a decreased level of yeast agglutination activity will be isolated. The strain carrying either Pfim-lacZ or Pmrk-lacZ will be also used as the recipient for the transposon-mediated mutagenesis, and the mutants showing alteration of LacZ activity will be isolated. After the insertion sequences determined, the genes of interest will be cloned and the specific gene deletion mutants generated, and the complementation assay will be carried out to confirm the regulatory activity. The mechanisms involved in the counter-expression regulation or interacting regulation between fimbriae and CPS will be further studied. 3) To investigate if envelope stress plays a role in the regulation of the counter-expression: An envelope stress resulted from the accumulation of MrkB, MrkC, MrkD, and MrkF at the periplasmic space could be built up in the mrkA deletion mutant. While in the mutant, expression of the type 1 fimbriae appeared to be switched on. Moreover, the rcsB deletion which reduced the CPS biosynthesis affected only the expression of type 1 fimbriae. These implied the changes of the envelope differentially affect the expression of type 1 or type 3 fimbriae. To determine if mrkA deletion leads to an envelope stress, expression of the regulatory genes involved in envelope stress responses will be analyzed by qRCR in the mrkA deletion mutant, and the mechanisms further characterized.