轉糖鏈球菌對酸，氧化及金屬離子壓力的基因調控

轉糖鏈球菌是一株經常在人類口腔中被發現的革蘭氏陽性兼性厭氧菌，其主要會造成牙齒的蛀蝕。自最初的菌落開始形成，轉糖鏈球菌必須對其所經歷連續的動態挑戰產生反應與適應，轉糖鏈球菌必須應付包括酸、營養物和必需元素的需求、巨噬細胞殺害等壓力。本篇研究重點在於轉糖鏈球菌面對金屬離子、弱酸與過氧化氫所產生的反應與基因調控。本研究的第一部分是關於口腔中的混合填充物會因腐蝕而釋出二價銅離子，發現二價銅離子會專一性的針對葡萄糖傳遞酶中的gtfD基因增加轉錄表現量，此調控與運送二價銅離子的copYAZ操縱子無關，顯示二價銅離子是調控gtfD基因的重要因子。再者發現酸對於葡萄糖傳遞酶的基因轉錄表現調控會受營養物的不同而改變，但卻不影響二價銅離子的調控。除了調控特定基因的表現之外，細菌還發展全面性的適應調控系統去面對一直變動的環境挑戰與助其生存於壓力環境中。轉糖鏈球菌面對弱酸的反應是本研究的第二部分。總體的調控系統可控制大量基因產生同步面臨環境壓力的表現，本部分之研究主要利用電腦和微陣列分析，發現受酸降低表現的操縱子中，GlnR box (ATGTNAN7TNACAT)位於轉糖鏈球菌參與胺基酸生合成與運送基因的控制子中，進一步發現參與胺基酸代謝的基因群會受到GlnR的負調控，而剔除此序列則使citBZC操縱子受酸減少轉錄表現的現象消失，因此結果顯示轉糖鏈球菌面對酸時會減少胺基酸前驅物的產生。已知三羧酸循環的中間代謝產物可經由專一的運送者輸入細菌體內，研究發現檸檬酸鹽的最高運送效率是經由CitM以質子電化學梯度驅動，利用一個質子與一個鈣離子-檸檬酸鹽複合物所形成電中性運送，結果顯示轉糖鏈球菌受外界檸檬酸鹽刺激可增加運送能力並增加在弱酸環境的生存能力，因此結果顯示檸檬酸鹽可調控轉糖鏈球菌的耐酸能力。第三部分的研究是關於轉糖鏈球菌對於過氧化氫的反應和基因調控。主要研究為轉糖鏈球菌如何保護自身對抗宿主之防禦系統並維持其於口腔中的生態。研究顯示，一對ScnR/ScnK雙組成系統可能參與調控細菌與細胞間作用機制。結果顯示無論野生株和突變株皆會被老鼠的巨噬細胞RAW 264.7吞噬，且巨噬細胞對於scnRK-null突變株的胞內敏感性增加。當活化的巨噬細胞吞噬野生株後，其活性氧的濃度明顯降低，但這樣的結果卻無法於突變株上觀察到，顯示由於中和活性氧的能力降低使得巨噬細胞的毒殺效果增加。此外，scnR-和scnRK-null對於過氧化氫的敏感性皆比野生株增加。有趣的是，scnRK基因的表現並不受過氧化氫所影響。轉糖鏈球菌的ScnRK基因在中和氧化壓力方面扮演一重要角色，並參與一部份抑制胞內活性氧生成而可減低被吞噬細胞毒殺之敏感性。

關鍵字

轉糖鏈球菌；二價銅離子；弱酸；過氧化氫

並列摘要

Streptococcus mutans is a Gram-positive, facultatively anaerobic bacteria commonly found in the human oral cavity and is a significant contributor to the caries on tooth. From initial colonization stages onward, S. mutans undergoes continuous dynamic challenges to which it must respond and adapt. S. mutans have to deal with stresses such as acidity, nutrients, essential elements, and the macrophage killing. The stress responses and gene regulation of S. mutans against metal ions, low pH and hydrogen peroxide (H2O2) were examined. In part I, copper ion (Cu2+) was released through corrosion of amalgam fillings in the oral cavity. The transcriptional expression of gtfD but not gtfB and gtfC was specifically induced by Cu2+ and independent of the Cu2+-transport operon copYAZ. Nutrient change influences the effect of pH not Cu2+. In addition to the expression regulation of specific genes, bacteria have evolved adaptive networks to face the challenges of a changing environment and to survive under conditions of stress. The response of S. mutans to low pH was investigated in the second part. The global regulatory systems control the simultaneous expression of a large number of genes in response to a variety of environmental stress factors. The GlnR box (ATGTNAN7TNACAT) was found in the promoter regions of acid-repressed operons involving in amino acid biosynthesis and transport in S. mutans based on microarray and in silico analyses. The expression of the amino acid metabolism clusters was negatively regulated by GlnR and deletion of this motif abolished the acidic repression of the citBZC operon. S. mutans reduced the production of amino acid precursors in response to acidity. The intermediate metabolites of TCA cycle could be specifically imported by transporters. The highest citrate transport rate of CitM was by the proton electrochemical gradient and supports an electroneutral transport mechanism with a coupling stoichiometry of one proton and one (Ca2+-citrate)1- complex. The citrate transport activity and survival of S. mutans at low pH are induced by the presence of citrate in the medium. The citrate modulates S. mutans aciduricity. Thirdly, the response and gene regulation of S. mutans to H2O2 was investigated. S. mutans avoids possible host defenses and maintain its ecological niche in the oral cavity. A putative two-component system (TCS), ScnR/ScnK, was invovled in the mechanisms underlying bacteria-cellular interaction. Both the wild-type and mutant strains were phagocytosed by murine macrophage RAW 264.7 cells at a comparable rate and an increased intracellular susceptibility was observed with the scnRK-null mutants. The amount of reactive oxygen species (ROS) in activated macrophages was reduced significantly after ingesting wild-type, but not scnRK-null mutant strains. This suggests that increased macrophage killing of these mutants is due to the impaired ability to counteract ROS. Additionally, both scnR- and scnRK-null mutants were more susceptible to H2O2. It is of interest to find that scnRK expression was unaffected by H2O2. ScnRK is important in counteracting oxidative stress in S. mutans. The decreased susceptibility to phagocytic killing is at least partly attributable to inhibition of intracellular ROS formation.

並列關鍵字

Streptococcus mutans ； copper ion ； low pH ； hydrogen peroxide

參考文獻

Abbe, K., Takahashi, S. & Yamada, T. (1982). Involvement of oxygen-sensitive pyruvate formate-lyase in mixed-acid fermentation by Streptococcus mutans under strictly anaerobic conditions. J Bacteriol 152, 175-182.

Ahmed, S. & Booth, I. R. (1983). The use of valinomycin, nigericin and trichlorocarbanilide in control of the protonmotive force in Escherichia coli cells. Biochem J 212, 105-112.

Akimaru, K., Utsumi, T., Sato, E. F., Klostergaard, J., Inoue, M. & Utsumi, K. (1992). Role of tyrosyl phosphorylation in neutrophil priming by tumor necrosis factor-alpha and granulocyte colony stimulating factor. Arch Biochem Biophys 298, 703-709.

Aoki, H., Shiroza, T., Hayakawa, M., Sato, S. & Kuramitsu, H. K. (1986). Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect Immun 53, 587-594.

Banas, J. A. & Vickerman, M. M. (2003). Glucan-binding proteins of the oral streptococci. Crit Rev Oral Biol Med 14, 89-99.

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轉糖鏈球菌對酸，氧化及金屬離子壓力的基因調控

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