Identification and Characterization of a Novel Serine Protease, VvpS, That Contains Two Functional Domains and Is Essential for Autolysis of Vibrio vulnificus
Identification and Characterization of a Novel Serine Protease, VvpS, That Contains Two Functional Domains and Is Essential for Autolysis of Vibrio vulnificus
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DOI：
10.1128/jb.00314-11
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发表时间：
2011-08-01
影响因子：
3.2
通讯作者：
Choi, Sang Ho
中科院分区：
文献类型：
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作者：
Lim, Moon Sub;Kim, Jeong-A;Choi, Sang Ho
革兰氏阴性菌自溶的分子机制知之甚少。在本研究中，我们确定了vvpS基因编码的丝氨酸蛋白酶，VvpS，创伤弧菌，革兰氏阴性食源性病原体。氨基酸序列预测VvpS由两个功能结构域组成，N-末端蛋白酶催化结构域（PCD）和C-末端碳水化合物结合结构域（CBD）。通过计数CFU和差异染色活细胞来测量，vvpS的无效突变显著增强了稳定期期间的活力。vvpS突变体减少了细胞质β-半乳糖苷酶和高分子量细胞外染色体DNA释放到培养上清液中，表明VvpS有助于创伤弧菌在稳定期的自溶。VvpS通过II型分泌系统（T2 SS）分泌，并且其在稳定期期间通过细胞内积累而对自溶发挥作用。与此一致，T2 SS的破坏加速了VvpS的细胞内积累，从而加速了创伤弧菌的自溶。VvpS还具有肽聚糖水解活性，表明创伤弧菌的自溶归因于VvpS对细胞壁的自我消化。通过C-末端缺失分析评估了VvpS结构域的功能，并证明PCD确实具有蛋白水解活性，并且CBD是有效水解肽聚糖所需的。最后，vvpS突变体在小鼠感染中表现出降低的毒力。结论：VvpS是一种具有模块化结构的丝氨酸蛋白酶，在创伤弧菌的自溶和致病过程中起重要作用。
Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus, a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic beta-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus. VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus.