克雷白氏肺炎桿菌中纖毛表現之分析

Abstract

克雷白氏肺炎桿菌是伺機性感染的革蘭氏陰性菌。利用生物資訊分析方法HMMER，我們在一株從台灣大學附設醫院的病患身上所分離出具有高致病性的NTUH-K0244菌株的基因體中找到九套線毛基因組。除了已被廣泛研究的第一型（fim）和第三型（mrk）線毛之外，其餘七套的功能以及特性都未曾被報導。我們分別將它們命名為kpa、kpb、kpc、kpd、kpe、kpf和kpg。為了了解各套線毛在特定條件下的表現情形，我們建構啟動子的活性分析系統。在各種測試條件下，第一型以及第三型線毛的啟動子活性都比其他七套要高。此外，這兩套線毛的活性在添加葡萄糖或甘油的培養環境下皆有顯著上升的情形。相對地，在高滲透壓力、過氧化物的存在，或偏鹼環境的刺激下，都造成活性的降低。 另一方面，我們在 kpd 基因組上游找到一個未知功能的基因，經由胺基酸序列比對（BLAST），我們認為其轉譯蛋白可能以雙分子訊息傳遞系統中調控子的角色來調控kpd基因組表現，因而命名為 kpdR。有趣的是，在克雷白氏肺炎桿菌中大量表現 KpdR蛋白時，我們發現beta-lactamase TEM-116蛋白明顯增加因而大大提高細菌對ampicillin的抵抗力；相對的，其生物膜的形成受到抑制。雖然，我們分別在CG43S3 和NTUH-K2044 兩株菌中可利用核酸增殖反應（PCR）得到 blaTEM-116，但經由比對現有的NTUH-K2044基因資料庫卻找不到該基因。今後，將確認 blaTEM-116的位置，並建構 kpdR 及blaSHV-1a的突變菌株，進一步了解 KpdR 與細菌對抗β-內酰胺類抗生素能力的關係。同時，我們在 kpb 及 kpf 基因組上游的位置分別找到類似的調控子基因，並分別命名為 kpbR 和 kpfR。而在克雷白氏肺炎桿菌中分別大量表現 KpbR 和 KpfR 蛋白時，我們發現細菌的生長都會受到抑制。 最後，我們發現移除座落於 mrk 及 fim 基因組之間的調控基因phgS 與 phgM，不僅會顯著降低mrk 啟動子的活性，kpg 線毛的啟動子活性也明顯下降；而進一步，分別在這兩個啟動子Pkpg和Pmrk片段中移除過去報導我們預測 PhgS 的辨認序列，原先受PhgS影響的現象已不復見，這些結果顯示PhgS和PhgM在調控kpg 與 mrk 線毛的表現上扮演特定的角色。

There are nine fimbiral operons identified in the genome of Klebsiella pneumoniae NTUH-K2044, a highly invasive strain isolated from Taiwan university hospital, by the HMMER search. The fim and mrk fimbriae had been described previously, but the others are novel fimbrial operons and are respectively named kpa, kpb, kpc, kpd, kpe, kpf, and kpg. The putative promoters of the nine operons were isolated, cloned in a LacZ reporter plasmid and the activities measured. In K. pneumoniae CG43S3Z01, the promoters of type 1 fimbriae (PfimA and PfimB) and type 3 fimbriae (PmrkA) had higher level of activity than those of the other fimbrial promoters. The activity of PfimA, PfimB, and PmrkA were enhanced under static cultures and the cultures addition with glucose or glycerol. On the other hand, the expression of fimB and mrkA were suppressed while the bacteria subject to higher osmotic pressure (＞200 mM NaCl), oxidative stress (60 □M H2O2), or the pH switch from 5.5 to 8. Interestingly, the overexpression of KpdR, a putative response regulator located upstream of the kpd gene cluster, appeared to decrease the biofilm formation and increase expression of beta-lactamase TEM-116 in both K. pneumoniae NTUH-K 2044 and CG43S3Z01. This implied a regulatory role of KpdR in the biofilm formation and also the resistance to the beta-lactam drug. Searching for the gene of blaTEM-116 and construction of the kpdR and blaSHV-1a mutants are in process. In the meantime, over-expressions of two other putative regulators, KpbR and KpfR, in K. pneumoniae appeared to interfere growth of the bacteria. Finally, the deletion of phgS or phgM was found to reduce not only the expression of type 3 fimbriae but also the activity of PkpgA. Truncation of the predicted consensus sequences for PhgS binding from PkpgA or PmrkA abolished the PhgS dependent expression. It was suggested that a regulatory role of PhgS for the expression of the two fimbriae.