重組豬乳鐵蛋白對克雷白氏肺炎桿菌CG43的抑菌功能研究

Abstract

摘要 乳鐵蛋白是一個78-80 kDa之攜鐵糖蛋白，主要分佈於哺乳動物的乳汁、唾液、眼淚及黏膜分泌液中。乳鐵蛋白不但具有廣泛的抑菌效果，還可以促進一些免疫細胞之增生，更有抑制癌細胞增生的能力。乳鐵蛋白經由胃蛋白酶水解所產生的N端片段，具有比完整蛋白更強之殺菌能力；此功能性片段稱為lactoferric -in (LFcin)，是乳鐵蛋白的主要抑菌區域。目前對於乳鐵蛋白及LFcin的抑菌機制並不明確，一般認為乳鐵蛋白可以與革蘭氏陰性菌外膜的酯多醣及孔隙蛋白結合，進而破壞細菌外膜的穩定而達到抑菌效果。為了了解乳鐵蛋白的抑菌機制，我們實驗室以大腸桿菌表現系統表現豬乳鐵蛋白的N端及C端片段 (N-LF, C-LF)，大量表現的結果卻使這些重組蛋白片段以包涵體 (inclusion body)方式堆積在細菌體內，我們以尿素使包涵體變性後再以梯度透析復性，仍無法獲得可溶性的蛋白質；我們改以大量適當濃度的氧化劑和還原劑稀釋蛋白質後，再以透析方式去除氧化劑、還原劑及尿素，終於獲得可溶性的重組蛋白。接著，我們將此重組蛋白定量加入紙錠，並以此紙錠分析重組蛋白的抑菌及攜鐵能力，結果證實這些再摺疊的重組蛋白具有活性。同時我們發現6.25□g N-LF可以抑制Klebsiella pneumoniae CG43及 Salmonella typhimurium LT2H1生長。另外，我們將K. pneumoniae CG43的全菌體蛋白以SDS-PAGE分離後，再以西方墨點法尋找可與此重組乳鐵蛋白結合的細菌蛋白，結果發現此菌的兩個主要膜蛋白可與乳鐵蛋白結合，而其中OmpK36孔隙蛋白 (porin)可能為乳鐵蛋白結合蛋白之ㄧ。我們接著以同源互換的方式建構K. pneumoniae CG43 ompK36突變株，再以乳鐵蛋白結合實驗比較野生株與此突變株之間的差異，確認OmpK36為K. pneumoniae CG43的乳鐵蛋白結合蛋白。再以disk assay 比較野生株與ompK36突變株之間的差異，我們發現重組豬乳鐵蛋白對野生株的抑菌作用較突變株明顯，這個現象可為乳鐵蛋白以K. pneumoniae CG43的孔隙蛋白作附著位置，進而破壞細胞膜以達到抑菌效果的假設提供了一個有利的證據。

Abstract Lactoferrin (LF) is a 78-80 kDa iron-binding glycoprotein which is present mostly in milk and other exocrine secretions. LF not only has a broad spectrum of antimicrobial properties, but also carries an activity to promote differentiation of granulocytes and proliferation of macrophage. Moreover, it has been reported that the bovine LF exhibited an inhibition effect on cancer growth. After digestion by pepsin, the LF releases a functional peptide, lactoferricin (LFcin), which possesses an even higher antimicrobial activity. Previous studies have shown that the antibacterial action resulted of the binding of LF and LFicin to LPS and porins of Gram-negative bacteria leads to alterations of the outer membrane stability. However, the exact antimicrobial mechanism of the LF and LFicin remains unclear. In order to clarify this mechanism, the plasmid clones encoding either the N-terminal or C-terminal regions of porcine lactoferrin, namely N-LF and C-LF, were expressed in E. coli, respectively. Unfortunately, these recombinant proteins formed inclusion bodies and the solubility couldn’t be improved even using urea to denature the proteins which were then refolded with gradient dialysis. Finally, the soluble proteins were obtained by using the protein refolding buffer which contains reduced and oxidized glutathione. The activities of these proteins were confirmed by using disk assay and iron utilization assays. A growth-inhibition activity of 6.25□g N-LF was found to either Klebsiella pneumoniae CG43 or Salmonella typhimurium LT2H1 using disc assay. The whole cell proteins of K. pneumoniae CG43 resolved in a SDS- polyacrylamide gel was subject to incubation with the purified proteins. Two LF binding proteins, presumably OmpK35 and OmpK36 of K. pneumoniae, were identified. Subsequently, an ompK36 mutant was constructed by homologous recombination. One of the LF-binding proteins was missing in the mutant demonstrated by western analysis indicated that OmpK36 is a LF binding protein. Growth inhibition activity of N-LF was also found to be decreased in the ompK36 mutant strain. Taken together, these results supported that the N-LF may bind to OmpK36 or other porins of K. pneumoniae CG43 and their binding leads to a growth inhibition.