行政院國家科學委員會專題研究計畫 期中進度報告
克雷白氏肺炎桿菌 DNA adenine methylase (Dam) 與致病
力相關性的研究(1/2)
計畫類別： 個別型計畫 計畫編號： NSC91-2314-B-002-178- 執行期間： 91 年 08 月 01 日至 92 年 07 月 31 日 執行單位： 國立臺灣大學醫學院內科 計畫主持人： 方啟泰 報告類型： 精簡報告 處理方式： 本計畫可公開查詢中 華 民 國 92 年 6 月 2 日
(一) 中文摘要 克雷白氏肺炎桿菌是一種革蘭氏陰性的腸內菌。在台灣，克雷白氏肺炎桿菌是最 重要的人類致病菌之一。常在有糖尿病的成人，甚至健康人，引起致命性的化膿 性肝膿瘍、菌血症、轉移性眼內炎及腦膜炎，此外克雷白氏肺炎桿菌也是一種重 要的院內感染病原菌，可造成肺炎，泌尿道感染。其致病力的調控機轉目前完全 不清楚。去氧核醣核酸腺核? 甲基化? 可把細菌基因體上核酸序列 GATC 的 adenine 甲基化，從而控制細菌基因表現，在細菌基因調控機轉上扮演重要角 色。在沙門氏桿菌-另一種革蘭氏陰性的腸內菌，去氧核醣核酸腺核? 甲基化? 在致病力調控機轉上的重要性已被證實，去氧核醣核酸腺核? 甲基化? 基因突變 之沙門氏桿菌菌株，致病力顯著下降。但去氧核醣核酸腺核? 甲基化? 在克雷白 氏肺炎桿菌致病力調控機轉的角色則尚未有人研究。本研究的目的即為探討去氧 核醣核酸腺核? 甲基化? 與克雷白氏肺炎桿菌致病力的相關性。我們目前已成功 的選殖出克雷白氏肺炎桿菌的去氧核醣核酸腺核? 甲基化? 基因。先利用由 NCBI (National Center for Biotechnology Information) 網站所搜尋到的 E. coli K12 的去氧核醣核酸腺核? 甲基化? 基因序列為模本，將其基因的 5’及 3’端序列設 計為引子對。以在臺灣已知為高毒力的克雷白氏肺炎桿菌原始型菌株 NTUH-2044 的基因體 DNA 為模板，用 PCR 的方式順利增幅出一段 669 個鹼基 的開放讀架片段。將此開放讀架片段定序後，與 E. coli K12 的去氧核醣核酸腺核 ? 甲基化? 基因序列作比對，發現有 99 ﹪的相似度。選殖出克雷白氏肺炎桿菌 菌株 NTUH-2044 的去氧核醣核酸腺核? 甲基化? 基因之後，我們進一步建構鑲 嵌有 chloramphenicol acetyltransferase (CAT) cassette 的去氧核醣核酸核? 甲基化 ? 基因，以 conjugation 的方法送回 NTUH-2044，篩選出 4 株 dam-基因突變菌株。
測試突變菌株的血清抗性，與野生型比較發現有明顯的降低。未來的研究將進而 以動物實驗測定其 50%致死量的變化，來量化去氧核醣核酸腺核? 甲基化? 基因 與克雷白氏肺炎桿菌致病力的相關性。進一步，將用微陣列晶片技術，比較原始 型菌株與突變型菌株在基因表現上的差異，來找出受到去氧核醣核酸腺核? 甲基 化? 基因控制的下游基因群。最後，將以動物實驗測試去氧核醣核酸腺核? 甲基 化? 基因突變菌株作為疫苗菌株的可行性。 關鍵詞：克雷白氏肺炎桿菌, 致病力, 去氧核醣核酸腺核? 甲基化?
(二) 英文摘要
Klebsiella pneumoniae, an enteric Gram-negative bacillus, is one of the most
important human pathogen in Taiwan. K. pneumoniae causes life-threatening pyogenic liver abscess, bacteremia, septic endophthalmitis and meningitis in both diabetic persons and otherwise healthy people. K. pneumoniae is also an important pathogen of nosocomial pneumonia and urinary tract infection. The regulation of virulence gene expression in K. pneumoniae has been poorly understood. DNA adenine methylase (Dam) can add methyl group to the adenine of sequence GATC, and thus play an important role in the control of bacterial gene expression. In
Salmonella, another enteric Gram- negative bacillus, the role of Dam in the
regulation of virulence has been well documented. dam- Salmonella mutants significantly loss the virulence potential. The role of Dam in K. pneumoniae has not yet been studied. The purpose of this proposal is to investigate the relationship between Dam and bacterial virulence in K. pneumoniae. We have successfully cloned the dam gene in K. pneumoniae. We used the nucleotides sequence of dam gene in E. coli K12, available on NCBI website, to design PCR primers. Using designed primers, we successfully amplify a DNA fragment containing a 669 bps open reading frame from the genomic DNA of clinical invasive strain K.
pneumoniae NTUH-2044. We further constructed dam- mutant by inserting chloramphenicol acetyltransferase (CAT) cassette into dam and sending the mutated
dam into wild type NTUH-2044 for homologous recombination. Four dam- K.
pneumoniae mutants were obtained, and all of them had much reduced serum
resistance in comparison with the wild type. In the second year, animal study will be used to determine the difference in LD50 to quantify the correlation between Dam and bacterial virulence in K. pneumoniae. Microarray technique will be used to identify the downstream genes regulated by dam. Finally, the possibility of dam- K.
pneumoniae mutants as vaccine strains will be explored through animal experiments.
(一) 前言
DNA 甲基化提供了細胞一套可以辨別外來 DNA 的機制，細菌的 DNA 甲基 化 的 角 色 通 常 被 認 為 可 保 護 自 身 的 DNA 免 於 受 到 限 制 內 切 ? (restriction endonuclease) 的 作 用 。 一 般 而 言 ， 負 責 DNA 甲 基 化 的 基 因 為 DNA methyltransferases。而 DNA methyltransferases 主要又分為 Dam(DNA adenine methyltransferase)以及 Dcm(DNA cytosine methyltransferase)，分別由 dam 及 dcm 基因所產生。在 E. coli 的 DNA 中 Dam 可以辨識 GATC 序列，並形成超過 99% 的 6-meAde。Dcm 則可辨認 CC(A/T)GG 序列，並形成全部的 5-meCyt。除了上 述這個角色之外，在 E. coli 中的研究發現，鹼基被甲基化之後還有其他的生物 功能。例如: 影響 DNA 的立體結構、dam-directed mismatch repair、改變 DNA 與 其調控蛋白質之間的親合力、調控染色體的複製起始以及調控基因的表現。
由於 Dam 在細胞生理上扮演許多重要的角色，所以在 dam 突變菌株上可 能會產生毒力減弱的現象。這種現象最早是在 E. coli 中發現，Dam 會調控 E. coli 的 pilus operons，而 pilus operons 對於 E. coli 引起的泌尿道感染及腹瀉扮演很重 要的角色。之後，陸續發現 Salmonella 的 Dam- 菌株變成無毒力，探究其原因可
能是基因表現失去了控制，才導致毒力消失。而在 Y. pseudotuberculosis 及 V.
cholerae 中 Dam 對其生存是必須的，但是在 Dam 過度表現的菌株中，其毒力也
是大大地降低。由此可見，Dam 扮演著調控病原菌與寄主之間的相互作用，並 對病原菌的毒力有所影響，但目前對 Dam 控制毒力的作用機制尚不清楚。 克雷白氏肺炎桿菌是一種革蘭氏陰性的腸內菌。在台灣，克雷白氏肺炎桿菌 是最重要的人類致病菌之一。常在有糖尿病的成人，甚至健康人，引起致命性的 化膿性肝膿瘍、菌血症、轉移性眼內炎及腦膜炎，此外克雷白氏肺炎桿菌也是一 種重要的院內感染病原菌，可造成肺炎，泌尿道感染。其致病力的調控機轉目前 完全不清楚。去氧核醣核酸腺核? 甲基化? 在克雷白氏肺炎桿菌致病力調控機轉 的角色則尚未有人研究。本研究的目的即為探討去氧核醣核酸腺核? 甲基化? 與 克雷白氏肺炎桿菌致病力的相關性。
（二）研究目的及文獻探討：
Klebsiella pneumoniae, an enteric Gram-negative bacillus, is one of the most
important human pathogen in Taiwan. K. pneumoniae causes life-threatening pyogenic liver abscess, bacteremia, septic endophthalmitis and meningitis in both diabetic persons and otherwise healthy people [111]. After 1995, K. pneumoniae replaces Streptococcus pneumoniae as the leading pathogen of adult community-acquired bacterial meningitis [2, 3]. Other common syndrome include pneumonia in patients with poorly controlled diabetes mellitus or alcoholism, and febrile neutropenia in patients received anticancer chemotherapy [1, 12]. K.
pneumoniae is also an important pathogen of nosocomial pneumonia, urinary tract
infection, bloodstream and wound infections [12]. Despite of in vitro effective antibiotic therapy, there is significant morbidity and an at least 10% mortality in patients with K. pneumoniae pyogenic liver abscess [411]. For patients with K.
pneumoniae meningitis, the mortality is as high as 30–40% despite in vitro effective
antimicrobial therapy [2, 3]. Further study of the pathogenesis of invasive K.
pneumoniae diseases in Taiwan is necessary in order to develop innovative
approaches for early diagnosis, more effective therapy, and possible prevention. Multiple bacterial virulent factors are probably involved during the dynamic process of invasive K. pneumoniae diseases, including factors promoting bacterial adhesion to epithelial surface, factors helping bacteria acquiring iron in tissue environment, factors protecting bacteria from attack of phagocytosis, complements and antibody, and factors that may directly damage host. Most previous studies on
K. pneumoniae pathogenesis were directed to one or more of the specific virulent
factors, such as polysaccharide capsule, lipopolysaccharide, adhesins, and iron siderophores [1222]. Since these virulence factors must act synergistically to provide bacteria the best chance of successful infection, there must be some regulatory mechanism to coordinate their expression. However, little was known about the global control mechanism of virulence gene expression in K. pneumoniae. DNA adenine methylase (Dam) plays a pivotal role in bacteria such as
Escherichia coli and Salmonella acting as a global regulator of gene expression
and affecting a wide range of critical cellular functions, including DNA replication, DNA repair, transposition, and segregation of chromosomal DNA [23]. This extraordinary versatility stems from the inherent biochemical activity of Dam. Thus, by adding methyl groups to various sties along the cellular DNA, Dam alters interactions of a variety of regulatory proteins with their designated gene targets and in the process, effectively controls expression of those genes. In some cases, such changes modulate bacterial virulence. Studies of E. coli pap operon have highlighted
the biological importance of Dam mechanism [24]. pap operon encode pili that enable E. coli to adhere to the urogenital tract and establish kidney infections. Genes encoding Pap pili are reversibly switched between the unexpressed state and the expressed state, a process known as phase variation. This ON-OFF pilin switching leads the bacteria to attach and detach, enable them first to colonize the bladder and then the kidney, causing cystitis and pyelonephritis, respectively. Pap phase variation is principally controlled by the leucin-responsive regulatory protein (Lrp) and the state of methylation of two specific pap regulatory DNA sequence that reside within Lrp-binding sites. During the OFF-phase, GATCprox is unmethylated, then Lrp binds to pap promoter proximal site and blocks pap transcription. During the ON-phase, when GATCprox is methylated, Lrp binding is blocked and pili are produced.
Studies in Salmonella, another enteric rod, also pointed out the importance of Dam in the regulation of bacterial virulence [25, 26]. Many genetically determined virulence functions of this pathogen are induced during infections but are not expressed, or expression is relatively low, when the cells are growing in vitro “On” in vivo, “OFF” in vitro. Because this response involves the coordinationed expression of may genes when cells are moved from host tissues to synthetic media, it suggests that the cells depend on global regulatory mechanism. Screening of mutants for the expression of in vivo induced (ivi) genes when cells are grown in vitro on syndthetic medium yielded cells with mutations that resulted in the loss of Dam activity. In turn, those changes derepress the activity of more than 35 distinct
ivi genes in Salmonella, indicating that Dam is a global regulator of Salmonella gene
expression. Salmonella strains that either lack Dam or overexpress it (DamOP) have severe virulence defects when tested in mice for their ability to cause typhoid fever. Mice survive and show no overt signs of disease when inoculated with 10,000-fold more Dam- or DamOP cells than are needed of the wild-type (Dam+) strain to kill 50% of the animals. Furthermore, inoculating mice with the Dam- mutants protected them when they were subsequently exposed to the virulent wild-type strain. The effectiveness of these attenuated mutants is somewhat surprising since other highly attenuated mutants typically are cleared too rapidly from animals to elicit protective responses.
Mutations in Dam and other adenine methyltransferase from an even wider variety of microbial pathogens, including Vibrio cholera, Yersinia pseudotuberculosis, can attenuate virulence, suggesting that enzymes which
methylate DNA are pivotal and might prove essential in bacterial pathogensis. However, the role of Dam in the virulence of K. pneumoniae has not yet been studied. The dam-equivalent gene in K. pneumoniae has not yet been sequenced and cloned, either. The purpose of this proposal is to investigate the relationship between
Dam and bacterial virulence in K. pneumoniae. （三）研究方法：
Bacterial strains, plasmids, and culture conditions
Clinical isolates of K. pneumoniae were collected at National Taiwan University Hospital (NTUH) from 1993 through 2000. The identification of the K. pneumoniae and the antibiotic susceptibility testing were performed according to standard clinical microbiologic methods [27] The K. pneumoniae strains were stored at -80 oC before use. K. pneumoniae NTUH-2044 was selected as the wild-type strain used in the subsequent study. The E. coli strains and most plasmids will be used in this study has been previously described [28]. Suicide vector pUTKm1ΔtnpΔblaΔmini-Tn5 [29, 30] has been constructed for the purpose of knockout. For general use, both E. coli and K. pneumoniae will be grown in Luria-Bertani (LB) broth or agar. When necessary, kanamycin (50 μg/ml) or chloramphenicol (50 μg/ml) will be added.
Cloning of Dam in K. pneumoniae
先利用由 NCBI (National Center for Biotechnology Information) 網站所搜尋 到的 E. coli K12 的 dam 基因序列為模本，將其基因的 5’及 3’端序列設計為引子 對。以在臺灣已知為高毒力的克雷白氏肺炎桿菌原始型菌株 NTUH-2044 的基因 體 DNA 為模板，用 PCR 的方式增幅出克雷白氏肺炎桿菌帶有 dam 基因序列的 DNA 片段，再接到 pGEM T-easy vector 上，以利後續實驗的進行。
DNA sequencing and analysis
DNA sequencing will be performed by using the ABI PRISM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Perkin- Elmer Applied Biosystems, Foster City, CA) on ABI PRISM 377 DNA Sequencer. Primers used for DNA sequencing will be purchased from Perkin-Elmer Biosystems. The nucleotide sequences and deduced amino acid sequences will be compared with those from nonredundant GenBank and SWISS-PROT databases by using computer software Sequencer 2.1.1 (Gene Codes Corp., Ann Arbor, MI) and the BLAST network service at the National Center for Biotechnology Information.
Knockout dam by homologous recombination
dam will be amplified by PCR , followed by TA cloning according to the recommended procedures provided by the manufacturer. A chloramphenicol acetyltransferase (CAT) cassette (gift from Dr. Diane Taylor) [33] will be inserted into dam on pGEM T-easy vector to construct cat:: dam. The cat:: dam fragment
will be further inserted into the EcoR1 site on suicide vector pUTKm1ΔtnpΔblaΔmini-Tn5. The knockout experiment will be carried out by sending linear DNA cat:: dam or a suicide plasmid vector
cat::cepA-pUTKm1ΔtnpΔblaΔmini-Tn5 into K. pneumoniae NTUH-2044 by
conjugation [2830], respectively. After conjugation, the recombinant clones will be selected on LB agar supplemented with chloramphenicol 50 μg/ml and ampicillin 100μg/ml to remove the donor E. coli S17-1/pir.
Serum resistant test
以 Podschun 等建立的方法來測試克雷白氏肺炎桿菌菌株的血清抗性強弱。 血清的來源由沒有感染過克雷白氏肺炎桿菌的健康成人志願捐出。測試菌量為 2.5 104 CFU/ml（colony-forming units）。將隔夜培養之克雷白氏肺炎桿菌菌液 以 LB 肉湯稀釋 100 倍，在 37C 搖晃培養 2–2.5 小時至菌液的吸光值在 600 nm （OD600）為 0.5。此時菌量約為 108 CFU/ml。將 OD600 0.5 的菌液以生理食鹽水 稀釋 100 倍。此時取 25 l 即為所需之接種菌量。將此 25 l 菌液與 75 l 人類血 清均勻混合後，於 37C 培養箱中培養，並在 0、1、2 及 3 小時時，分別以序列 稀釋法，將不同倍數的稀釋液（以生理食鹽水稀釋）均勻塗布於 LB 培養基上。 隔夜培養後，計算培養基上菌落數目。克雷白氏肺炎桿菌之血清抗性以存活菌數 百分比（survival ratio）來表示。 (四) 目前成果 1. 克雷白氏肺炎桿菌 dam gene 選殖:
我們已成功的利用由 NCBI (National Center for Biotechnology Information) 網站 所搜尋到的 E. coli K12 的 dam 基因序列為模本，將其基因的 5’及 3’端序列設計 為引子對。以在臺灣已知為高毒力的克雷白氏肺炎桿菌原始型菌株 NTUH-2044 的基因體 DNA 為模板，用 PCR 的方式順利增幅出一段 669 個鹼基的開放讀架 片段。選殖出克雷白氏肺炎桿菌菌株 NTUH-2044 的 dam 基因。將此開放讀架片 段定序後，與 E. coli K12 的 dam 基因序列作比對，發現有 99 ﹪的相似度。 2. dam-突變菌株的建構:
建構鑲嵌有 chloramphenicol acetyltransferase (CAT) cassette 的去氧核醣核酸核? 甲基化? 基因，以 conjugation 的方法送回 NTUH-2044，篩選出 4 株 dam-基因突
變菌株。 Cont rol dam - 4 dam - 3 dam - 2 dam - 1 wt
Fig1. 以 PCR 來檢測是否得到 dam- 的突變菌株，wt 為 wild type NTUH-2044，而在突變菌株可檢測到鑲嵌有 chloramphenicol acetyltransferase (CAT) cassette 的去氧核醣核酸腺核? 甲基化? 基因。 3. dam-突變菌株的血清抗性測試: 以 Podschun 等建立的方法來測試克雷白氏肺炎桿菌菌株的血清抗性強弱。血清 的來源由沒有感染過克雷白氏肺炎桿菌的健康成人志願捐出。測試菌量為 2.5 104 CFU/ml（colony-forming units）。將隔夜培養之克雷伯氏肺炎桿菌菌液以 LB 肉湯稀釋 100 倍，在 37C 搖晃培養 2–2.5 小時至菌液的吸光值在 600 nm（OD600） 為 0.5。此時菌量約為 108 CFU/ml。將 OD600 0.5 的菌液以生理食鹽水稀釋 100 倍。此時取 25 l 即為所需之接種菌量。將此 25 l 菌液與 75 l 人類血清均勻混 合後，於 37C 培養箱中培養，並在 0、1、2 及 3 小時時，分別以序列稀釋法， 將不同倍數的稀釋液（以生理食鹽水稀釋）均勻塗布於 LB 培養基上。隔夜培養 後，計算培養基上菌落數目。克雷白氏肺炎桿菌之血清抗性以存活菌數百分比 （survival ratio）來表示。畫出其關係圖(Fig.2 )，可明顯看出突變菌株的血清抗 性下降。 Ratio Strain 0hr 1hr 2hr 3hr KP wt 1 1.6 1.79 5.9 dam- 1 1 0.81 0.63 0.91 dam- 2 1 0.4 0.4 0.47 dam- 3 1 0.87 1.07 0.75 dam- 4 1 0.89 1.2 1.22 1kb
Fig.2 克雷白氏肺炎桿菌之血清抗性之存活菌數百分比，以 0 小時為基準，及以 此結果所得到的線性圖。由 serum resistant test 的結果發現，突變株對 serum 的 抵抗能力下降。
(五) 討論
先前的研究指出 dam 會影響許多病原細菌的致病性(pathogenesis)及生長， 而且 Dam 可以調控基因表現。因此，在 dam 突變後，Klebsiella pneumoniae 是 否會產生毒力減弱的現象；以及哪些基因的表現發生改變，而這些基因及可能與
Klebsiella pneumoniae 的致病機轉或生長有關。所以本實驗則著重於研究 DNA
adenine methyltransferase 對於克雷伯氏肺炎桿菌致病性產生的影響。 未來的研究將進而以動物實驗測定其 50%致死量的變化，來量化去氧核醣核 酸腺核? 甲基化? 基因與克雷白氏肺炎桿菌致病力的相關性。進一步，將用微陣 列晶片技術，比較原始型菌株與突變型菌株在基因表現上的差異，來找出受到去 氧核醣核酸腺核? 甲基化? 基因控制的下游基因群。最後，將以動物實驗測試去 氧核醣核酸腺核? 甲基化? 基因突變菌株作為疫苗菌株的可行性。 (六)參考文獻
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（七）計畫成果自評
完成本計畫第一年預定之進度:
1. 選殖到克雷白氏肺炎桿菌菌株 NTUH-2044 的 dam 基因並完成定序的工 作,dam 基因長度為 669 個鹼基。與 E. coli K12 的 dam 基因序列作比對，發現 有 99 ﹪的相似度。
2. 建構鑲嵌有 chloramphenicol acetyltransferase (CAT) cassette 的去氧核醣核酸 核? 甲基化? 基因，以 conjugation 的方法送回 NTUH-2044，篩選出 4 株 dam
-基因突變菌株。 3. 測試克雷白氏肺炎桿菌之去氧核醣核酸腺核? 甲基化? 基因突變的菌株的 血清抗性可明顯看出突變菌株的血清抗性下降。因此，在 dam 突變後，克雷白 氏肺炎桿菌是否會產生毒力減弱的現象；以及哪些基因的表現發生改變，而這 些基因及可能與克雷白氏肺炎桿菌的致病機轉有關。有待進一步的研究。 目前初步的研究結果尚未完備，dam 基因在克雷白氏肺炎桿菌的致病機轉 上，扮演何種角色及其相關性，都有待進一步的研究來釐清。未來的研究將進而 以動物實驗，來量化去氧核醣核酸腺核? 甲基化? 基因與克雷白氏肺炎桿菌致病 力的相關性。進一步，將用微陣列晶片技術，比較原始型菌株與突變型菌株在基 因表現上的差異，來找出受到去氧核醣核酸腺核? 甲基化? 基因控制的下游基因 群。最後，將以動物實驗測試去氧核醣核酸腺核? 甲基化? 基因突變菌株作為疫 苗菌株的可行性。
