Fur蛋白質在克雷白氏肺炎桿菌CG43中的功能探討

Abstract

在細菌生命週期中，具有足量的鐵離子是維持新陳代謝和生長必需的，因此多數細菌演化出同時擁有數個螯鐵系統。然而，過多鐵離子卻會造成氧化自由基形成，這對細菌本身將會造成致命的傷害，是故微生物體內的螯鐵系統都需要受到緊密的調控。在革蘭氏陰性菌中，許多參與螯鐵系統基因轉錄過程都受到Fur 蛋白質的抑制。此外,除了調控鐵離子濃度在細菌體內外的平衡，當細菌遭受到酸性或過氧化環境壓力Fur 蛋白質也參與啟動保護機制。克雷白氏肺炎桿菌CG43是一具有毒性的臨床分離株，被K2血清型莢膜所包覆。為了瞭解Fur在克雷白氏肺炎桿菌CG43中扮演的角色，我們建構了fur基因缺損株，此基因缺損明顯影響細菌生長。有趣的是，fur突變株菌落相較於野生株顯得比較黏稠，透過莢膜多醣定量顯示fur突變株莢膜明顯增量；進一步分析啟動子活性，我們發現莢膜多醣合成基因組orf3-15表現量因fur基因缺損而增加；而fur突變株對小白老鼠的半致死劑量較野生株減少，顯示Fur蛋白質可能參與調控細菌的毒性。另外，我們也發現fur缺損使克雷白氏肺炎桿菌CG43對於酸性(pH 3)和過氧化(H2O2 treatment)的環境壓力變得較為敏感。最後，fur的缺損還會提高iro、iuc基因組(攝取三價鐵相關)以及feo基因組(攝取二價鐵相關)的啟動子活性，顯示Fur 蛋白質在調控克雷白氏肺炎桿菌體內鐵離子平衡上扮演重要的角色。

Bacteria have evolved several acquisition systems for sufficient quantities of iron to support their metabolism and growth. Iron overloading would lead to the formation of hydroxyl radicals and hence microorganisms have equipped a tight regulatory system for iron uptake. In Gram-negative bacteria, Fur protein represses the transcription of many genes that are involved in iron acquisition. In addition to control iron homeostasis, Fur also participates in protective responses to acid stress and oxidative stress. Herein, we report the construction of fur deletion mutant and found that the deletion impaired the growth of Klebsiella pneumoniae CG43S3, a highly virulent strain heavily encapsulated with K2 serotype. Interestingly, the deletion rendered the bacteria more mucoid phenotype which probably resulted from increasing amount of the glucuronic acid content. In addition, an increased activity of Pcps-orf3-15 was found. The deletion of fur slight reduced the LD50 using mouse lethality assay suggesting an involvement of Fur in virulence regulation. Moreover, fur deletion was found to increase the bacterial sensitivity to either acidic or oxidative stress. Finally, promoter activity measurement revealed that the fur deletion enhanced the activity of iro and iuc, respectively encoding enterobactin and aerobactin siderophore uptake systems, and feoABC, coding for ferrous iron uptake system. This indicated that Fur plays a major role for the regulation of iron acquisition system in the bacteria.