克雷白氏菌侵入細胞相關蛋白IalK的功能研究

Abstract

摘要 克雷白氏肺炎桿菌 (Klebsiella pneumoniae) 是一株引起院內感染僅次於大腸桿菌的伺機性病原菌，常引起小兒、老年人和免疫能力較差的宿主肺炎、尿道感染及膿瘍等病癥。我們實驗室先前以聚合脢連鎖反應由高毒性的克雷白氏菌CG43中選殖了一段可能與致病相關的核酸序列，這段核酸序列與Bartonella bacilliformis IalA (invasion-associated protein A) 的胺基酸序列有40 %的一致性，因此命名為IalK。為了探討IalK在克雷白氏肺炎桿菌致病過程中扮演的角色：首先，我們利用大腸桿菌表現系統在低溫下大量表現IalK蛋白質，再經純化濃縮獲得足夠量可溶性的IalK，並證明其dinucleoside polyphosphatase的酵素活性。藉由受質專一性的活性測試，我們發現Gp3G為最佳受質，Km值為8.6 μM。我們進一步從克雷白氏肺炎桿菌CG43噬菌體基因庫中篩選出包含ialK基因片段的噬菌體，用聚合脢連鎖反應選殖建構ialK基因缺損的質體，再以接合作用送入克雷白氏菌CG43S3，經由同源互換的方式得到ialK缺損的突變株。而利用細胞模式比較克雷白氏菌野生株和 IalK突變株的差異性，我們發現ialK突變株和野生株對Int-407均有黏附能力，但是對Int-407和Hep-2並無明顯的侵入作用。我們同時發現，雖然ialK突變株的生長速率明顯降低，但對小白鼠Balb/c的毒性沒有明顯的影響。最後，可能因為IalK在菌體表現量太低或多株抗體的專一性不夠，我們無法偵測IalK在克雷白氏肺炎桿菌中確切的位置。綜合以上的結果，我們推測IalK藉由分解人類血小板釋放出的Gp3G來干擾宿主內的生理活性，以達成在細胞內存活的目的。

Abstract Klebsiella pneumoniae is an opportunistic pathogen that often causes septicemia and urinary tract infections in immunocompromised patients. We have previously isolated from a virulent K. pneumoniae strain CG43 an invasin-like gene ialK, which has a 40% amino acid sequence identity with the Bartonella bacilliformis invasion-associated locus ialA. Sequence analysis revealed that the IalK as well as the IalA contain a conserve motif of the dinucleoside polyphosphatase family. The ialK has been hererologously expressed in Escherichia coli and the recombinant protein purified. Dinucleoside polyphosphatase activity of the purified IalK has been demonstrated using a group of dinucleoside polyphosphate including Ap3A, Ap4A, Ap5A, Ap6A, Gp3G, Gp4G, Gp5G as the substrate, respectively. Gp3G was found to be the best substrate for recombinant IalK with a Km of 8.6 μM, which is comparable with that of the B. bacilliformi IalA using Ap4A as the substrae. We have constructed an ialK mutant of K. pneumoniae by alleic exchange through homologous recombination. There was no difference detected in adherence to Int-407 cells between the wild type strain and the ialK mutant, however. No invasion activity was shown even in the wild type strain K. pneumoniae CG43 using both Int-407 and Hep-2 cell lines as the assay model, indicating a suitable cell line has yet been obtained. Although, the mutant exhibited a decreasing growth rate in a minimal medium, no significant affect on its virulence could be measured as determined by a mice peritonitis model. Finally, the polyclonal antibody prepared by immunizing mice with the purified IalK failed to determine the cellular location of the IalK in K. pneumoniae awaits the preparation of a more specific antibody. In conclusion, the Gp3G hydrolase activity of IalK may function to reduce the stress-induced dinuceoside polyphosphatate levels during invasion and so enhance the survival of the pathogen.