Identification and characterization of a putative magnesium transport associated gene mgtC in Klebsiella pneumoniae

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。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.口試委員會審定書.................................................. Ⅰ謝.............................................................. Ⅱ文摘要.......................................................... Ⅲ文摘要.......................................................... Ⅴ一章、緒論.......................................................1二章、材料與方法.................................................5.克雷伯氏肺炎桿菌菌株........................................5.聚合酶鏈鎖反應 (polymerase chain reaction, PCR) .................5.萃取克雷伯氏肺炎桿菌 total RNA.............................. 6.克雷伯氏肺炎桿菌基因微陣列 .................................7.反轉錄定量聚合酶鏈鎖反應 (RT-qPCR) ........................ 10.建構克雷伯氏肺炎桿菌基因剔除株 ............................12.建構ΔmgtC突變株的染色體互補株(cis-complementation) .........13.不同鎂離子濃度NCE培養液生長觀察 .........................14.利用穿透式電子顯微鏡觀察線毛表現 ..........................150.利用光學顯微鏡 (light microscope)觀察菌體變長情形 ........... 151.測量血清中鎂離子的濃度.....................................162.動物實驗...................................................16三章、 結果......................................................20.利用克雷伯氏肺炎桿菌基因微陣列尋找在糖尿病人血清條件下表現量上升的基因.................................................20.利用real-time PCR 確認微陣列結果，確定mgtC基因在糖尿病人血清條件下表現量上升...........................................20.mgtC基因在臨床菌株中的分布比例............................21.比較NTUH-K2044野生株在鎂離子濃度10mM、1.8mM、0.7mM與10μM NCE培養液中mgtC基因的RNA表現量........................21.利用RT-PCR確認mgtC基因及其下游KP2872基因為同一個操縱組 (operon) ................................................... 22.NTUH-K2044野生株及 ΔmgtC基因剔除株KP2872基因 RNA表現量.........................................................22.比較NTUH-K2044野生株及ΔmgtC基因剔除株隔夜生長情形......23.利用穿透式電子顯微鏡 (transmission electron microscopy, TEM) 觀察ΔmgtC基因剔除株在鎂離子濃度10mM、1.8mM、0.7mM與10μM NCE培養液中表型 (phenotype) 的改變.............................23.比較NTUH-K2044野生株及ΔmgtC基因剔除株在鎂離子濃度10mM、1.8mM與0.7mM NCE培養液中菌體變長的情形................. 240.建構NTUH-K2044ΔmgtC::mgtC染色體互補株 (cis-complementation)並觀察其表型.................................................241.利用克雷伯氏肺炎桿菌基因微陣列尋找mgtC基因在鎂離子濃度10mM與10μM 環境下所調控的基因................................252.NTUH-K2044野生株、ΔmgtC基因剔除株fim及mrk基因表現量情形.........................................................263.NTUH-K2044野生株、ΔmrkA基因剔除株fimD基因表現量情形....264.ΔmrkA fimA基因剔除株、ΔmgtC fimA基因剔除株、ΔmgtC mrkA基因剔除株、ΔmgtC mrkA fimA基因剔除株在鎂離子濃度10mM、1.8mM與0.7mM NCE培養液下之線毛表現情形......................... 275.血清中鎂離子濃度的測量.................................... 276.探討NTUH-K2044野生株及ΔmgtC基因剔除株對小鼠的致病力... 28四章、 總結與討論................................................30五章、 參考文獻..................................................70目錄一、研究中使用的細菌菌株及載體....................................36二、實驗中使用到的引子........................................... 38三、利用克雷伯氏肺炎桿菌基因微陣列工具，以NTUH-K2044野生株培養在LB培養液為對照組、NTUH-K2044野生株培養在含有50%糖尿病人血清之LB培養液中為實驗組，比較兩者基因的RNA表現量.............40四、以PCR觀察PLA (pyogenic liver abscess) 與non-PLA strains之mgtC基因的分布.......................................................42五、比較NTUH-K2044野生株及各種克雷伯氏肺炎桿菌基因剔除株在鎂離子濃度10mM及10μM NCE minimal medium生長下型態上的改變.......43六、利用光學顯微鏡 (microscope) 觀察在鎂離子濃度10mM、1.8mM及0.7mM環境下NTUH-K2044野生株、 ΔmgtC基因剔除株及ΔmgtC::mgtC染色體互補菌株菌體變長的情形並計算菌體長度.........................44七、國家動物中心引進美國The Jackson Laboratory第二型糖尿病鼠品種和人類第二型糖尿病症狀與基因背景的比較...........................45八、以腹膜腔注射及胃內感染的方式，比較NTUH-K2044野生株及ΔmgtC基因剔除株對於BALB/cByl、C57BL/6J小鼠及B6.V-Lepob/J第二型糖尿病鼠的致病力...................................................46九、以腹膜腔注射的方式，比較NTUH-K2044野生株及ΔmgtC基因剔除株在C57BL/6J小鼠及B6.V-Lepob/J和BKS.Cg-m +/+ Leprdb/J第二型糖尿病鼠體內的競爭情形...............................................47十、利用克雷伯氏肺炎桿菌基因微陣列工具，以NTUH-K2044野生株培養在鎂離子濃度10μM NCE minimal medium為對照組、ΔmgtC基因剔除株培養在鎂離子濃度10μM NCE minimal medium為實驗組，比較兩者基因的RNA表現量..................................................48十一、利用穿透式電子顯微鏡 (transmission electron microscopy, TEM) 觀察NTUH-K2044野生株及各克雷伯氏肺炎桿菌基因剔除株線毛表現情形.........................................................49目錄一、利用克雷伯氏肺炎桿菌基因微陣列比較培養在LB培養液之NTUH-K2044野生株與培養在50% 糖尿病血清的LB培養液之NTUH-K2044野生株mRNA表現量的差異...........................................50二、利用Real-time PCR比較NTUH-K2044野生株在LB培養液及含有50%糖尿病人血清之LB培養液中mgtC基因的RNA表現量...............51三、 (A) 利用NCBI BLAST比較克雷伯氏肺炎桿菌、Salmonella enterica和Salmonella typhimurium此三株菌之mgtC基因序列相似度，利用CLUSTALW找出最具保守性的基因序列設計引子 (MgtC-56F、MgtC-511R) 。 (B) NTUH-K2044野生株染色體上mgtC (KP 2873) 及附近基因相對位置和方向。.......................................52四、利用Real-time PCR比較NTUH-K2044野生株在不同鎂離子濃度10mM、1.8mM、0.7mM、10µM NCE培養液中mgtC基因的RNA表現量........54五、利用RT-PCR確認mgtC基因及其下游KP2872基因為同一個操縱組 (operon)......................................................55六、NTUH-K2044野生株、ΔmgtC基因剔除株和ΔmgtC::mgtC染色體互補株在鎂離子濃度10μM NCE培養液中mgtC基因的RNA表現量，NTUH-K2044野生株和ΔmgtC基因剔除株在鎂離子濃度10mM、1.8mM與0.7mM NCE培養液中KP2872基因的RNA表現量..............................56七、pKO3-Km基因剔除載體及突變株篩選過程.........................57八、pKO3-Km基因互補載體及染色體互補株篩選過程...................58九、在鎂離子濃度10mM、1.8mM、0.7mM與10μM NCE培養液中，比較 NTUH-K2044野生株和ΔmgtC基因剔除株生長情形................59十、觀察NTUH-K2044 野生株、ΔmgtC基因剔除株及ΔmgtC::mgtC染色體互補菌株在鎂離子濃度10mM及10μM環境下細菌聚集 (autoaggregation) 的現象.......................................................60十一、利用穿透式電子顯微鏡 (TEM) 觀察菌體型態改變................61十二、利用穿透式電子顯微鏡 (TEM) 觀察NTUH-K2044 野生株、ΔmgtC基因剔除株及NTUH-K2044 ΔmgtC::mgtC染色體互補株在鎂離子濃度10μM環境下線毛 (fimbriae)表現......................................62十三、克雷伯氏肺炎桿菌培養在鎂離子濃度10μM NCE培養液下NTUH-K2044野生株與ΔmgtC基因剔除株mRNA表現量的差異..................64十四、NTUH-K2044野生株和ΔmgtC基因剔除株、 NTUH-K2044野生株和ΔmrkA基因剔除株在鎂離子濃度1.8mM、0.7mM NCE培養液中mrkA與fim基因的RNA表現量............................................66十五、利用穿透式電子顯微鏡 (TEM) 觀察NTUH-K2044野生株及各基因剔除株在鎂離子濃度 10mM 及 1.8mM、0.7mM NCE培養液下生長的線毛 (fimbriae) 表現...............................................67十六、 健康人、第二型糖尿病人及健康小鼠、ob/ob和db/db第二型糖尿病鼠血清中鎂離子濃度的比較.......................................68十七、 糖尿病小鼠以胃內感染的方式餵食NTUH-K2044野生株與ΔmgtC基因突變株的存活比例與時間圖...................................6