Analysis of proline reduction in the nosocomial pathogen Clostridium difficile
Analysis of proline reduction in the nosocomial pathogen Clostridium difficile
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DOI：
10.1128/jb.01370-06
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发表时间：
2006-12-01
影响因子：
3.2
通讯作者：
Self, William T.
中科院分区：
文献类型：
--
作者：
Jackson, Sarah;Calos, Mary;Self, William T.
艰难梭菌是一种具有蛋白水解作用的严格厌氧菌，近年来已成为临床上重要的医院病原体。发病机制是由于产生致命毒素A和B，即大梭菌细胞毒素家族的成员。虽然已经确定生长培养基中氨基酸含量的改变会影响毒素的产生，但这种观察到的影响的分子机制尚不清楚。由于缺乏这种病原体所使用的氨基酸发酵途径的信息，我们研究了Stickland反应是否可能是其生物能量途径的核心。艰难梭菌在Stickland对上的生长在限制性基础培养基中产生细胞密度的大幅增加，表明这些反应与ATP产生相关。硒的补充是必需的，这种增加细胞产量。基因组序列数据的分析揭示了编码两种关键硒酶还原酶，甘氨酸还原酶和D-脯氨酸还原酶（PR）的蛋白质组分的基因。这些硒酶表达后，添加相应的Stickland受体（甘氨酸，脯氨酸，或羟脯氨酸）。硒酶D-脯氨酸还原酶的纯化揭示了PrdA和PrdB（含SeCys）蛋白质的混合复合物。PR只利用D-脯氨酸，但不L-羟脯氨酸，即使在表达和纯化的脯氨酸消旋酶的存在下。PR被认为是独立的二价阳离子，锌是一个有效的抑制剂PR。这些结果表明，Stickland反应是艰难梭菌生长的关键，PR的机制可能与以前研究的PR从非致病性物种显着不同。
Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and D-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.