創傷弧菌(Vibio vulnificus) 屬於革蘭氏陰性病原菌，藉由傷口或含菌的食物感染人類，會造成敗血症和厲害的皮膚病變如壞死性筋膜炎。此菌一個重要的毒力因子是multifunctional-autoprocessing RTX (MARTX)，具細胞毒殺性，也能保護細菌免於被吞噬細胞吞噬。過去我們意外分離到一株失去運動能力、細胞毒殺性和對小鼠毒力的菌株NY303。此菌株於可轉譯出白胺酸轉錄調節子(leucine-responsive regulatory protein, lrp) 的基因上發生了自發性點突變；將lrp 基因剔除可得到和NY303 類似之表型特性，顯示Lrp 可能參與在創傷弧菌之毒力調控機制中。為了瞭解lrp 突變株如何失去細胞毒殺性，我們首先確認MARTX 的mRNA和蛋白質表現量，發現在NY303 和lrp 突變株中，不管是rtxA1 之mRNA或MARTX 之蛋白質產量都減少了。負責將MARTX 由細胞內分泌至細胞外之基因，rtxB、rtxD 和rtxE 的mRNA 表現量也下降了。進一步觀察被lrp 突變株感染的RAW264.7 細胞，也發現其所含之MARTX 的量較被野生株感染的細胞少。但是，雖然突變株之MARTX 表現量減少，卻還具有抑制吞噬細胞吞噬的能力，故推測在lrp 突變株中，MARTX表現量降低可能並非造成其失去對小鼠的毒力之主要原因。為了瞭解Lrp 如何調控創傷弧菌之毒力，我們測試細菌lrp 突變株之移生能力。相較於野生株，lrp 突變株在一般小鼠背部氣囊之移生能力極差，而且當感染嗜中性白血球低下之小鼠，或是將野生株和lrp 突變株共同感染一般小鼠，lrp 突變株之移生能力只能部分恢復，故推測lrp 突變株進到小鼠體內時會面臨生長的缺陷，導致其毒力之喪失。而我們也發現lrp 突變株在小鼠血清中的生長較野生株差。但有趣的是，lrp 突變株在小鼠全血中的生長較小鼠血清中好，故我們推測紅血球中的血紅素可以幫助lrp 突變株的生長。的確，當於小鼠血清中加入氯化血紅素(hemin)、血紅蛋白(hemoglobin)，或是可產生游離態三價鐵離子之檸檬酸鐵銨(ferric ammonium citrate)，皆可使lrp 突變株的生長能力恢復。而lrp 突變株中和鐵質獲取相關之基因，如和siderophore 生合成相關之基因或血紅素接受器HupA之mRNA表現量也較野生株低，由此結果推測lrp 突變株攝鐵能力較差可能是導致此突變株進入宿主後無法正常地生長的原因之一。

Vibrio vulnificus, a gram-negative bacterial pathogen, can cause septicemia and skin lesions as severe as necrotizing fasciitis via wound or food-borne infection. An important virulence factor of this organism is the multifunctional-autoprocessing RTX (MARTX), which mediates cytotoxicity and can protect the bacteria from phagocytosis. We have serendipitously isolated a spontaneous avirulent mutant, NY303, with greatly reduced motility/chemotaxis, cytotoxicity and virulence in the mouse. The spontaneous mutation resulting in loss of cytotoxicity and virulence was mapped to the gene encoding leucine-responsive regulatory protein (lrp). A mutant with a deletion in lrp (lrp mutant) displayed a phenotype similar to that of NY303, confirming the involvement of Lrp in the regulation of virulence. In this study, we checked the mRNA and protein levels of MARTX in NY303 and the lrp mutant, and found that they were reduced in both mutants. However, the ability of the mutants to inhibit phagocytosis was not affected, suggesting that the reduced MARTX levels may not be responsible for loss of virulence of the lrp mutants. We further found that the lrp mutant colonized poorly in the mouse due to its defect in growth per se after entering the mouse body. The poor growth of lrp mutant in mouse serum was greatly enhanced in that supplemented with hemin, hemoglobin or ferric ammonium citrate, and a few iron acquisition-associated genes were down-regulated in this mutant. The defect in iron acquisition may partly explain the poor growth of lrp mutant in vivo.

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