克雷伯氏肺炎桿菌鎂離子傳送相關mgtC基因之選殖與功能研究

近二十年來，一種新型的侵襲性克雷伯氏肺炎桿菌逐漸成為全球性的重要社區感染病原菌，會引發細菌性肝膿瘍 (pyogenic liver abscess, PLA) 合併菌血症及轉移性眼內炎或腦膜炎，糖尿病人在社區感染中佔了45-75% 的比例。本實驗室之前利用克雷伯氏肺炎桿菌基因微陣列，尋找野生株在糖尿病人血清的刺激條件下表現量有差異的基因，經過real-time PCR確認，mgtC為表現量上升的基因之一，本研究發現mgtC基因分布情形在PLA菌株較non-PLA菌株為高 (98% vs.75%)，具有統計上的意義 (P = 0.004，chi-square test)。ΔmgtC基因剔除株在鎂離子濃度1.8mM、0.7mM的NCE培養液中，生長的情形和野生株並沒有差異，但利用穿透式電子顯微鏡 (transmission electron microscopy, TEM) 觀察ΔmgtC基因剔除株發現有菌體變長 (cell elongation) 且細菌聚集 (autoaggregation) 的現象，另外觀察到纖毛 (fimbriae) 的表現。利用微陣列找到ΔmgtC基因突變株在低鎂NCE培養液條件下第一型 (fim) 及第三型 (mrk) 線毛基因表現量上升，推測mgtC基因可能扮演抑制線毛表現的角色。利用穿透式電子顯微鏡觀察ΔmgtC mrkA、ΔmgtC fimA、ΔmrkA fimA及ΔmgtC mrkA fimA基因剔除株線毛的表現，發現ΔmgtC mrkA及ΔmgtC fimA基因剔除株皆有線毛的表現，但ΔmrkA fimA及ΔmgtC mrkA fimA基因剔除株皆沒有線毛的表現。過去文獻報告，糖尿病人血清鎂離子濃度較健康人低，因此目前推測，mgtC基因可能可以在低鎂濃度的糖尿病人血清中，有利於克雷伯氏肺炎桿菌致病因子的表現，初步以C57BL/6J老鼠腹膜腔或胃內感染野生株或ΔmgtC基因剔除株，半致死劑量沒有差異，而以腹膜腔或胃內感染B6.V-Lepob/J第二型糖尿病鼠，半致死劑量也沒有差異。進一步以腹膜腔注射方式同時感染野生株及ΔmgtC基因剔除株觀察在糖尿病鼠內競爭的情形，發現ΔmgtC基因剔除株競爭力為野生株的3倍，同樣以腹腔注射同時感染野生株及ΔmgtC mrkA fimA基因剔除株，競爭只略強於野生株，ΔmgtC mrkA fimA基因剔除株競爭力為野生株的2倍。綜上所述，利用穿透式電子顯微鏡觀察剔除株線毛表現與否，與小鼠活體內競爭情形，證實線毛的表現可能為第一型 (fim) 或第三型 (mrk) 線毛單一型表現抑或是兩者同時表現，有待進一步實驗證實。

關鍵字

克雷伯氏肺炎桿菌；第二型糖尿病； mgtC ；第一型線毛；第三型線毛； fim gene cluster ； mrk gene cluster

並列摘要

In the past 20 years, community-acquired pyogenic liver abscess (PLA) caused by a new type of invasive Klebsiella pneumoniae has become a global emerging disease. Some patients develop serious complications such as bacteremia, metastatic meningitis or endophthalmitis. Diabetes mellitus (DM) is a predisposing condition, with a prevalence ranging from 45% to 75% in patients with K. pneumoniae liver abscess. In the previous study, we used a K. pneumoniae microarray to compare transcriptional profiles between wild-type strains and ΔmgtC mutants induced by the DM serum. The microarray data were confirmed by real-time polymerase chain reaction (real-time PCR), and we found mgtC gene expression was increased. In this study, we found mgtC was significantly more prevalent in PLA strains. The growth of the ΔmgtC mutants were similar to the growth of wild-type strains in the NCE liquid medium supplemented with 1.8mM or 0.7mM Mg2+. The ΔmgtC strains showed autoaggregated bacteria in 10μM Mg2+ medium. Morphology analysis evaluated by TEM indicated that ΔmgtC mutants exhibited cell elongation as well as fimbriae expression in 1.8mM、0.7mM Mg2+ medium. Therefore, we used a K. pneumoniae microarray to compare transcriptional profiles between wild-type strains andΔmgtC mutants induced by 10μM Mg2+ medium. We discovered that type 1 (fim) and type 3 (mrk) fimbriae genes expression were increased in ΔmgtC mutants grown in 10μM Mg2+ medium by using microarray analysis. These results suggested that mgtC may play a role in a repressor system for those fimbriae expression. Fimbriae expression of ΔmgtC mrkA, ΔmgtC fimA double mutants or ΔmgtC mrkA fimA triple mutants was examined by TEM. ΔmgtC mrkA and ΔmgtC fimA double mutants produced fimbriae, but ΔmgtC mrkA fimA triple mutants had no fimbriae extended. Hypomagnesemia has been reported to occur among type 2 DM patients. It was indicated that mgtC gene may induce the virulence factors of K. pneumoniae to express when K. pneumoniae was exposed to low diabetic serum Mg concentration. There was no difference in LD50 between wide-type strains and ΔmgtC mutants by i.g. or i.p. infected C57BL/6J and B6.V-Lepob/J mice. We coinfected wide-type strains with ΔmgtC mutants by i.p. to investigate which is more competitive in type 2 DM mice. ΔmgtC mutant out competed the wild type strain about 3-fold. Competition of ΔmgtC mrkA fimA mutants and wide-type strains in vivo indicated that ΔmgtC mrkA fimA mutant was about 2 times competitive than wild-type strains but weaker than ΔmgtC mutant. These data suggested that either or both type 1 (fim) and type 3 (mrk) gene expressed for fimbriae production.

並列關鍵字

Klebsiella pneumoniae ； type Ⅱ diabetes mellitus ； mgtC ； type 1 fimbriae ； type 3 fimbriae ； fim gene cluster ； mrk gene cluster

參考文獻

15. Barton, E.N., et al., Diabetes mellitus and Klebsiella pneumoniae liver abscess in adults. Trop Geogr Med, 1991. 43(1-2): p. 100-4.

2. Wang, J.H., et al., Primary liver abscess due to Klebsiella pneumoniae in Taiwan. Clin Infect Dis, 1998. 26(6): p. 1434-8.

3. Ko, W.C., et al., Community-acquired Klebsiella pneumoniae bacteremia: global differences in clinical patterns. Emerg Infect Dis, 2002. 8(2): p. 160-6.

4. Chang, S.C., et al., Klebsiella pneumoniae isolates causing liver abscess in Taiwan. Diagn Microbiol Infect Dis, 2000. 37(4): p. 279-84.

5. Yang, C.C., et al., Comparison of pyogenic liver abscess caused by non-Klebsiella pneumoniae and Klebsiella pneumoniae. J Microbiol Immunol Infect, 2004. 37(3): p. 176-84.

國際替代計量

克雷伯氏肺炎桿菌鎂離子傳送相關mgtC基因之選殖與功能研究

全文下載

主題瀏覽